Method for producing protein

ABSTRACT

It has been believed that promoting the assembly of polysomes composed of many ribosomes attached to mRNA is very effective for highly efficient protein synthesis. However, the mechanism for p180 protein&#39;s capability of promoting polysome formation has been yet to be elucidated. 
     The inventors of the present application newly discovered SF3b4 protein as a protein that specifically interacts with the coiled-coil domain of p180 protein, a responsible region for its capability of promoting polysome formation, and which is capable of promoting mRNA localization to an endoplasmic reticulum (ER). The inventors also found that, in cells capable of highly expressing both p180 protein and a protein promoting mRNA localization to an endoplasmic reticulum (ER) (e.g., SF3b4 protein), the mRNA localization to the endoplasmic reticulum can be significantly elevated so that the secretory capacity in cultured cells can be enhanced. Further, the inventors demonstrated that when a particular nucleotide sequence is inserted into an expression plasmid, SF3b4 protein exhibiting protein expression enhancing ability can be localized onto the endoplasmic reticulum membrane, and the mRNA distribution in polysomes can be shifted towards heavier fractions, whereby the secretory capacity in cells can be enhanced.

This application is a continuation of U.S. National Phase applicationSer. No. 14/780,313 which has a 371(c) date of Sep. 25, 2015, now U.S.Pat. No. 9,884,897, which is based on International Application No.PCT/JP2014/058702 filed Mar. 26, 2014 (published as WO2014/157429 onOct. 2, 2014) which claims the benefit of Japanese Applications2013-064357 filed Mar. 26, 2013 and 2013-261178 filed Dec. 18, 2013,each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

This application includes as part of its disclosure a biologicalsequence listing text file which is being submitted via EFS-Web. Saidbiological sequence listing is contained in the file named“49416o1001.txt” having a size of 57,570 bytes that was created Feb. 4,2019, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present application relates to a recombinant cell for enhancingprotein expression from an exogenous gene in recombinant cells, as wellas an invention using such a cell. More specifically, this applicationrelates to providing a cell having enhanced expression of p180 proteinand/or SF3b4 protein, or a method for enhancing a synthetic or secretorycapacity of a protein as a product of interest and consequentlyproducing a protein, with the use of a cell having such characteristics.The method for enhancing a synthetic or secretory capacity of a proteinas a product of interest and consequently producing a protein, with theuse of a cell having such characteristics, is also characterized byusing a cis-element in a vector for expressing the protein as a productof interest.

BACKGROUND ART

In the field of biotechnological pharmaceuticals developed by applyinggenetic recombination technologies, particularly the market for antibodypharmaceuticals has grown rapidly in recent years while there have beenraised concerns about their loading on medical expenses; so there hasalways been a demand for developing a technique for producingbiotechnological pharmaceuticals that enable more efficient proteinproduction and are more low-cost than conventional ones.

Examples of hosts that have been used for protein production usinggenetic recombination technologies include animal cells, yeast, andEscherichia coli. E. coli and the like are capable of producing aprotein as a product of interest with low costs, but are unfit forglycoprotein production because no post-translational modification suchas sugar-chain modification can be achieved in such microorganisms. Inaddition, E. coli has a tendency to form an inclusion body containing aproduced protein, and thus has a disadvantage in that, in order toobtain a protein as a product of interest, a solubilization process isfurther required after synthesis, thereby causing heavy workload.

Particularly in the case of glycoproteins such as antibodies, an addedsugar chain has an influence on the water solubility of a protein as aproduct of interest, its resistance to a protease, its tissue-targetingcapability, and its biological activity; thus, there has been a need forproduction technologies using animal cells from higher eukaryotes, andthese technologies have advanced considerably in recent years. Underthese circumstances, many current antibody pharmaceuticals are producedusing Chinese hamster ovary (CHO) cells, and optimizing productionprocesses for such pharmaceuticals is still an important challenge.

Proteins secreted extracellularly from eukaryotic cells includingmammalian cells are synthesized in the endoplasmic reticulum which isintracellular organelle divided by endomembranes. The endoplasmicreticulum is broadly classified into the following two types: a roughendoplasmic reticulum studded on its surface with ribosomes which aremachines for protein synthesis composed of a RNA-protein macrocomplex,and a smooth endoplasmic reticulum with no ribosomes, but the detailedmechanism of formation of the rough endoplasmic reticulum has been yetto be elucidated.

In the living body, there are professional secretory cells specializedin secreting particular proteins, and these professional secretory cellshave highly developed rough endoplasmic reticulum which are consideredto enable highly efficient protein production. Examples of suchprofessional secretory cells include fibroblasts secreting collagen, andpancreatic exocrine secretory cells secreting a group of digestiveenzymes. As compared to those professional secretory cells, roughendoplasmic reticulum such as CHO cells and HEK293 cells, which are nowoften used for genetically engineered protein production, areproblematic in that they are present only in a very small amount and areinferior in secretory activity.

In the process of production of biotechnological pharmaceuticals usinggenetic recombination technologies, the genes of a protein as a productof interest are under the control of a promoter showing hightranscription activity in an expression vector, and are presumed toexpress their mRNA at a high level. However, even under theseconditions, the mRNA level is often not correlated with the expressedprotein amount per se, and one of the factors for this may be due to lowefficiency of mRNA translation on the endoplasmic reticulum membranes.

These observation suggest that there may be room for further enhancementof the protein synthetic capacity in the aforementioned cells that arenow widely used for genetically engineered protein production, if mRNAcan be provided in a more appropriate manner to be used to the machinesfor translation on the endoplasmic reticulum membranes like in the caseof fibroblasts.

It is known that fibroblasts permanently secreting collagen constantlyexpress a high level of collagen protein-encoding mRNAs, the majority ofwhich is detected on the endoplasmic reticulum, a place of biosynthesisof the secretory proteins (Non-patent Literature 1). However, its morelocalization of the collagen mRNA on the endoplasmic reticulum is notsufficient to activate collagen synthesis, but the formation of apolysome having high translation efficiency is also needed for activatedsynthesis.

The previous analyses made by the present inventors revealed that themRNAs for some types of proteins, including collagen genes, have atendency to form a polysome in which multiple ribosomes, machines forprotein synthesis are associated to each other (Patent Literature 1,Non-patent Literature 2). This finding led to the conjecture that thereason why, in the process of production of biotechnologicalpharmaceuticals using genetic recombination technologies, genetranscripts encoding a protein of interest are expressed at a high leveland nevertheless the protein is synthesized or secreted only in a smallamount, it may be because in used cells, mRNA is not provided to themachines for translation on the endoplasmic reticulum membranes in aneasy-to-use form.

CITATION LIST Non-Patent Literatures

Non-patent Literature 1: Ueno, T., et al., (2010). J Biol Chem 285 (39),29941-50.

Non-patent Literature 2: Ueno, T., et al., (2012). Regulation ofpolysome assembly on the endoplasmic reticulum by a coiled-coil protein,p180. Nucleic Acids Res.

Patent Literature

Patent Literature 1: Japanese Patent Application Publication No. JP2011-227462

SUMMARY OF INVENTION Technical Problem

For the purpose of protein synthesis on the endoplasmic reticulum, thelocalization of mRNA to the endoplasmic reticulum (polysome formation)is indispensable. Further, it has been believed that promoting theformation of polysomes composed of many ribosomes attached to mRNA isvery effective for highly efficient protein synthesis. However, themechanism for p180 protein's capability of promoting polysome formationhas been yet to be elucidated.

Solution to Problem

The inventors of the subject application have made in-depth analysis,and as a result newly discovered SF3b4 (splicing factor 3B subunit 4)protein as a protein that specifically interacts with the coiled-coildomain of p180 protein, a responsible region for promotion of polysomeformation (Non-patent Literature 2), to thereby promote mRNAlocalization to the endoplasmic reticulum (ER). Further, the inventorscreated cells capable of highly expressing both SF3b4 protein and p180protein, and as a result found that, in those cells having such acharacteristic, the mRNA localization to the endoplasmic reticulum canbe significantly elevated so that the secretory capacity in culturedcells can be enhanced. Thus, the inventors have completed the presentinvention.

The present inventors demonstrated that there can be provided arecombinant cell having enhanced intracellular expression of the fulllength or a portion of p180 protein and enhanced intracellularexpression of a protein promoting mRNA localization to an endoplasmicreticulum (ER), and having enhanced intracellular synthetic or secretorycapacity of a protein as a product of interest.

The present inventors also demonstrated that, in the second mode of thepresent invention, there can be provided a method in which, in arecombinant cell having enhanced expression of the full length or aportion of p180 protein and enhanced expression of a protein promotingmRNA localization to an endoplasmic reticulum (ER), a nucleic acidmolecule encoding a protein as a product of interest is transformed orproduction of the protein as a product of interest is increased, wherebybiosynthetic or secretory capacity of the protein as a product ofinterest is enhanced and consequently the protein as a product ofinterest is produced.

It was demonstrated that the present invention can solve theaforementioned problems by providing such a characteristic recombinantcell, or by using said characteristic recombinant cell and enhancing thesynthetic or secretory capacity of a protein as a product of interest.

[1] A recombinant cell having enhanced intracellular expression of thefull length or a portion of p180 protein, or a protein promoting mRNAlocalization to an endoplasmic reticulum (ER), or both of said proteins,and having enhanced intracellular synthetic or secretory capacity of aprotein as a product of interest.

[2] The recombinant cell as set forth in [1], wherein the p180 proteinis selected from the group consisting of:

(a) a protein that consists of an amino acid sequence with at least 70%sequence identity to the amino acid sequence (SEQ ID NO: 2) ofhuman-derived p180 protein, and which has a capability of promotingpolysome formation on an intracellular endoplasmic reticulum membrane;

(b) a protein that consists of an amino acid sequence derived from theamino acid sequence (SEQ ID NO: 2) of the human-derived p180 protein bydeletion, substitution, or addition of one or more amino acids, andwhich has a capability of promoting polysome formation on theintracellular endoplasmic reticulum membrane;

(c) a protein that consists of an amino acid sequence specified by anucleotide sequence with at least 70% sequence identity to thenucleotide sequence (SEQ ID NO: 1) of a gene encoding the human-derivedp180 protein, and which has a capability of promoting polysome formationon the intracellular endoplasmic reticulum membrane;

(d) a protein that consists of an amino acid sequence specified by anucleotide sequence derived from the nucleotide sequence (SEQ ID NO: 1)of the gene encoding the human-derived p180 protein by deletion,substitution, or addition of one or more nucleotides, and which has acapability of promoting polysome formation on the intracellularendoplasmic reticulum membrane; and

(e) a protein that consists of an amino acid sequence specified by anucleotide sequence hybridizable under stringent conditions with anucleotide sequence complementary to the nucleotide sequence (SEQ IDNO: 1) of the gene encoding the human-derived p180 protein, and whichhas a capability of promoting polysome formation on the intracellularendoplasmic reticulum membrane.

[3] The recombinant cell as set forth in [1] or [2], wherein the p180protein is derived from a mammalian animal.

[4] The recombinant cell as set forth in [3], wherein the full length orthe portion of the mammalian p180 protein is the human p180 protein (SEQID NO: 2), murine p180 protein (GenBank Accession No. NP_077243), ratp180 protein (GenBank Accession No. XP_230637), Chinese hamster p180protein (GenBank Accession No. XM_003496471), canine p180 protein(GenBank Accession No. NP_001003179), equine p180 protein (GenBankAccession No. XP_001915027), simian p180 protein (GenBank Accession No.XP_002798281), chimpanzee p180 protein (GenBank Accession No.XP_514527), porcine p180 protein (GenBank Accession No. XP_001926148),or a portion thereof.

[5] The recombinant cell as set forth in any one of [1] to [4], whereinthe portion of the p180 protein is selected from: a portion comprisingan amino acid sequence corresponding to a region consisting of the aminoacids at positions 27 to 157 of a protein having the amino acid sequenceof SEQ ID NO: 2 (human p180 protein); a portion comprising an amino acidsequence corresponding to a region consisting of the amino acids atpositions 623 to 737 of said protein; a portion comprising an amino acidsequence corresponding to a region consisting of the amino acids atpositions 738 to 944 of said protein; and a portion comprising an aminoacid sequence corresponding to a region consisting of the amino acids atpositions 945 to 1540 of said protein.

[6] The recombinant cell as set forth in any one of [1] to [5], whereinthe protein promoting mRNA localization to the endoplasmic reticulum(ER) is selected from the group consisting of the full length or aportion of splicing factor 3B subunit 4 (SF3b4) protein (the full-lengthamino acid sequence 424 AA of SEQ ID NO: 4; RRM1 consisting of 13 to 91AA of SEQ ID NO: 4; and RRM2 consisting of 100 of SEQ ID NO: 4).

[7] The recombinant cell as set forth in [6], wherein the SF3b4 proteinis selected from the group consisting of:

(i) a protein that consists of an amino acid sequence with at least 70%sequence identity to the amino acid sequence (SEQ ID NO: 4) ofhuman-derived SF3b4 protein, and which has a capability of promotingmRNA localization to the endoplasmic reticulum;

(ii) a protein that consists of an amino acid sequence derived from theamino acid sequence (SEQ ID NO: 4) of the human-derived SF3b4 protein bydeletion, substitution, or addition of one or more amino acids, andwhich has a capability of promoting mRNA localization to the endoplasmicreticulum;

(iii) a protein that consists of an amino acid sequence specified by anucleotide sequence with at least 70% sequence identity to thenucleotide sequence (SEQ ID NO: 3) of a gene encoding the human-derivedSF3b4 protein, and which has a capability of promoting mRNA localizationto the endoplasmic reticulum;

(iv) a protein that consists of an amino acid sequence specified by anucleotide sequence derived from the nucleotide sequence (SEQ ID NO: 3)of the gene encoding the human-derived SF3b4 protein by deletion,substitution, or addition of one or more nucleotides, and which has acapability of promoting mRNA localization to the endoplasmic reticulum;and

(v) a protein that consists of an amino acid sequence specified by anucleotide sequence hybridizable under stringent conditions with anucleotide sequence complementary to the nucleotide sequence (SEQ ID NO:3) of the gene encoding the human-derived SF3b4 protein, and which has acapability of promoting mRNA localization to the endoplasmic reticulum.

[8] The recombinant cell as set forth in [6] or [7], wherein the SF3b4protein is derived from a mammalian animal.

[9] The recombinant cell as set forth in [8], wherein the full length orthe portion of mammalian SF3b4 protein is the human SF3b4 protein (SEQID NO: 4), murine SF3b4 protein (GenBank Accession No. NP_694693.1), ratSF3b4 protein (GenBank Accession No. NP_001011951.1), Chinese hamsterSF3b4 protein (GenBank Accession No. XP_003498680.1), canine SF3b4protein (GenBank Accession No. XP_540295.3), equine SF3b4 protein(GenBank Accession No. XP_001488649.2), simian SF3b4 protein (GenBankAccession No. NP_001097793.1), chimpanzee SF3b4 protein (GenBankAccession No. XP_513768.2), porcine SF3b4 protein (GenBank Accession No.XP_001926524.1), or a portion thereof.

[10] The recombinant cell as set forth in any one of [1] to [9], whereinthe synthetic or secretory capacity of the protein as a product ofinterest is enhanced by transforming a nucleic acid molecule encodingthe protein as a product of interest or increasing production of theprotein as a product of interest.

[11] A cell line designated by Accession No. NITE BP-01753 (CHO 3D5),Accession No. NITE BP-1535 (CHO YA7), or Accession No. NITE ABP-01811(CHO 1B2).

[12] A method for producing a protein as a product of interest, wherein,in a recombinant cell having enhanced expression of the full length or aportion of p180 protein, a protein promoting mRNA localization to anendoplasmic reticulum (ER), or both of said proteins, a nucleic acidmolecule encoding the protein as a product of interest is transformed orproduction of the protein as a product of interest is increased, wherebythe protein as a product of interest is produced.

[13] The method as set forth in [12], wherein the p180 protein isderived from a mammalian animal.

[14] The method as set forth in [13], wherein the full length or theportion of the mammalian p180 protein is human p180 protein (SEQ ID NO:2), murine p180 protein (GenBank Accession No. NP_077243), rat p180protein (GenBank Accession No. XP_230637), Chinese hamster p180 protein(GenBank Accession No. XM_003496471), canine p180 protein (GenBankAccession No. NP_001003179), equine p180 protein (GenBank Accession No.XP_001915027), simian p180 protein (GenBank Accession No. XP_002798281),chimpanzee p180 protein (GenBank Accession No. XP_514527), porcine p180protein (GenBank Accession No. XP_001926148), or a portion thereof.

[15] The method as set forth in [13] or [14], wherein the portion of themammalian p180 protein is selected from: a portion comprising a regionconsisting of the amino acids at positions 27 to 157 of a protein havingthe amino acid sequence of SEQ ID NO: 2 (human p180 protein); a portioncomprising a region consisting of the amino acids at positions 623 to737 of said protein; a portion comprising a region consisting of theamino acids at positions 738 to 944 of said protein; and a portioncomprising a region consisting of the amino acids at positions 945 to1540 of said protein.

[16] The method as set forth in any one of [12] to [15], wherein theprotein promoting mRNA localization to the endoplasmic reticulum (ER) isselected from the group consisting of the full length or a portion ofsplicing factor 3B subunit 4 (SF3b4) protein (the full-length amino acidsequence 424 AA of SEQ ID NO: 4; RRM1 consisting of 13 to 91 AA of SEQID NO: 4; and RRM2 consisting of 100 of SEQ ID NO: 4).

[17] The method as set forth in [16], wherein the SF3b4 protein isderived from a mammalian animal.

[18] The method as set forth in [17], wherein the full length or theportion of mammalian SF3b4 protein is the human SF3b4 protein (SEQ IDNO: 4), murine SF3b4 protein (GenBank Accession No. NP_694693.1), ratSF3b4 protein (GenBank Accession No. NP_001011951.1), Chinese hamsterSF3b4 protein (GenBank Accession No. XP_003498680.1), canine SF3b4protein (GenBank Accession No. XP_540295.3), equine SF3b4 protein(GenBank Accession No. XP_001488649.2), simian SF3b4 protein (GenBankAccession No. NP_001097793.1), chimpanzee SF3b4 protein (GenBankAccession No. XP_513768.2), porcine SF3b4 protein (GenBank Accession No.XP_001926524.1), or a portion thereof.

[19] The method as set forth in any one of [12] to [18], wherein therecombinant cell is a cell line designated by Accession No. NITEBP-01753 (CHO 3D5), Accession No. NITE BP-1535 (CHO YA7), or AccessionNo. NITE ABP-01811 (CHO 1B2).

[20] The method as set forth in any one of [12] to [19], wherein theprotein as a product of interest is a glycoprotein.

[21] The method as set forth in [20], wherein the protein as a productof interest is a collagen, a fibronectin, or an antibody.

[22] A method for increasing an amount of a protein as a product ofinterest to be expressed in a cell as an expression system, wherein, inan expression unit for expressing the protein as a product of interest,a cic-element to be recognized/bound (or interacted with) by anRNA-binding protein is inserted, downstream of a promoter and upstreamof the start codon in the nucleotide sequence of a DNA encoding theprotein as a product of interest, whereby the amount of the protein as aproduct of interest to be expressed in the cell as an expression systemis increased.

[23] The method as set forth in [22], wherein the cis-element is to berecognized/bound (or interacted with) by an RNA recognition motif(RRM)-type RNA-binding protein.

[24] The method as set forth in [23], wherein the cis-element is to berecognized/bound (or interacted with) by an RNA recognition motif (RRM)of the SF3b4 protein.

[25] The method as set forth in any one of [22] to [24], wherein thecis-element has a nucleotide sequence containing one or more 9mer to12mer sequence motifs GAN-(X)_(n)-ACN₂ (n=3 to 6) (N₁ and N₂ can beindependently any of the nucleotides A, T, C and G).

[26] The method as set forth in [25], wherein the cis-element has anucleotide sequence containing one or more 9mer to 12mer sequence motifs(GAG-(X)_(n)-ACV (n=3 to 6) (V represents A, G or C), SEQ ID NOs: 17 to20).

[27] The method as set forth in any one of [22] to [26], wherein thenucleotide sequence of the cis-element is a sequence selected from thegroup consisting of: a sequence derived from the nucleotide sequence ofthe 5′ untranslated region of a type I collagen gene; a sequence derivedfrom the nucleotide sequence of the 5′ untranslated region of afibronectin gene; a sequence derived from the nucleotide sequence of the5′ untranslated region of the matrix metalloproteinase 14 (MMP14) gene;a sequence derived from the nucleotide sequence of the 5′ untranslatedregion of the prolyl 4-hydroxylase A2 (P4HA2) gene; and a sequencederived from the nucleotide sequence of the 5′ untranslated region ofthe prolyl 4-hydroxylase A1 (P4HA1) gene.

[28] The method as set forth in any one of [22] to [27], wherein thenucleotide sequence of the cis-element is any sequence selected from thegroup consisting of the full length of SEQ ID NO: 5 or the full lengthof SEQ ID NO: 7, and the nucleotides at positions 1 to 102, positions 1to 78, positions 1 to 60, positions 61 to 126, positions 16 to 57,positions 79 to 126, positions 103 to 126, positions 58 to 78, positions51 to 78, positions 1 to 27, and positions 70 to 78 of SEQ ID NO: 5.

[29] The method as set forth in any one of [22] to [28], wherein thecell as an expression system is an intact host cell, a cell havingenhanced expression of the full length or a portion of p180 protein, acell having enhanced expression of the full length or a portion of SF3b4protein, or a cell having enhanced expression of both of said proteins.

[30] A medicinal composition for suppressing collagen synthesis andpreventing the alveolar epithelium and aggravation of fibrosis due toabnormal collagen through functional inhibition or expressionsuppression of SF3b4.

Advantageous Effects of Invention

It was found that, by means of using the recombinant cell of the presentinvention which has enhanced expression of the full length or a portionof p180 protein and/or a protein promoting mRNA localization to theendoplasmic reticulum (ER) (e.g., the full length or a portion of SF3b4protein), as well as transforming a DNA encoding a protein as a productof interest, a synthetic or secretory capacity of the protein as aproduct of interest is dramatically enhanced, and consequently theprotein as a product of interest is produced efficiently. It is alsodemonstrated that when a cis-element is added into an expression unit,SF3b4 protein that exhibits protein expression enhancing ability can belocalized onto an endoplasmic reticulum, and the mRNA distribution inpolysomes can be shifted towards heavier fractions, whereby thesecretory capacity in the cells can be enhanced.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the results of Western blotting analysis of the differenttypes of cells prepared in Example 1 (CHO cells, CHO 3D5 cells, CHO 5 gcells, and CHO YA7 cells) for intracellular expression of p180 proteinas well as SF3b4 protein, a protein promoting mRNA localization to anendoplasmic reticulum (ER).

FIG. 2 comparatively shows the results of expression of secretedalkaline phosphatase (SEAP) protein in the cases of exogenouslyintroducing an expression plasmid for human placental SEAP as asecretory marker into each of the different types of cells prepared inExample 1.

FIG. 3 comparatively shows the degrees of localization of secretedalkaline phosphatase (SEAP) mRNAs to the membrane fractions in the casesof exogenously introducing an expression plasmid for human placentalSEAP as a secretory marker into each of the different types of cellsprepared in Example 1.

FIGS. 4A-C shows a schematic diagram of a position for insertion of acis-element-containing expression vector (FIG. 4A), and the results ofevaluating cis-element's secretion activation capability (FIG. 4B andFIG. 4C).

FIG. 5 shows variations in protein secretory activity in the cases wherecollagens were expressed.

FIG. 6 shows that antibody secretion is activated by cis-elementinsertion.

FIG. 7 shows a comparison of the effects of the kozak sequence andcis-element #1 on secretion activation, using the CHO cells and the CHOYA7 cells.

FIG. 8 shows the relationship of cis-element structure with proteinexpression enhancement effect, using cis-element #1, cis-element #2,cis-element #3, and cis-element #4.

FIG. 9 shows that an example of the motif in a cis-element isGAG-(X)_(n)-ACN₂ (n=3 to 6) (A), and also shows a diagram for evaluatingvarious elements for SEAP secretory activity (B).

FIG. 10 shows an investigation of the influences of substitutions,deletions, or insertions of nucleotides in the motif GAN₁-(X)_(N)-ACN₂on the motif activity (FIG. 10A and FIG. 10B).

FIG. 11 shows that collagen production is remarkably suppressed throughsuppression of SF3b4 expression.

FIG. 12 shows that the COL1A1 cDNA weight in polysome fraction shiftedtowards higher density fractions in the presence of a cis-element ascompared to the absence of a cis-element.

DESCRIPTION OF EMBODIMENTS

The present inventors demonstrated that, in the first mode of thepresent invention, there can be provided a recombinant cell havingenhanced intracellular expression of the full length or a portion ofp180 protein and/or the full length or a portion of splicing factor 3Bsubunit 4 (SF3b4) protein, and having enhanced intracellular syntheticor secretory capacity of a protein as a product of interest.

In this mode of the present invention, the full length or a portion ofp180 protein and/or the full length or a portion of SF3b4 protein are/iscaused to be expressed in a cell, thereby making it possible to promotepolysome formation on an intracellular endoplasmic reticulum, which isinvolved by mRNA, an expression product of a nucleic acid moleculeencoding a protein as a product of interest. As referred to herein, thepolysome refers to a complex in which multiple ribosomes on theintracellular endoplasmic reticulum are bound to one mRNA molecule. Suchup-regulated polysome formation results in an enhancement of abiosynthetic or secretory capacity of a protein as a product ofinterest, thereby enabling production of the protein.

The aforementioned recombinant cell of the present invention is firstlycharacterized by having enhanced intracellular expression of the fulllength or a portion of p180 protein, particularly mammalian p180protein. The p180 protein refers to an essential endoplasmic reticulummembrane protein which is abundantly expressed in secretory tissues andcapable of promoting polysome formation.

In this connection, it is known that as compared to human p180 protein(GenBank Accession No. AB287347), murine p180 protein has 87% similarityin amino acid sequence, rat p180 protein has 87% similarity, Chinesehamster p180 protein has 88% similarity, canine p180 protein has 91%similarity, equine p180 protein has 89% similarity, simian p180 proteinhas 91-92% similarity, chimpanzee p180 protein has 98% similarity, andporcine p180 protein has 86% similarity. It is reported that the aminoacid identities of all mammalian p180 proteins fall within a range notlower than 84%, and that even those of the p180 proteins of other livingorganisms fall within a range not lower than 76%.

TABLE 1 Sequence homologies of p180 of various species to human p180Protein similarity Gene to identity human to (%): human amino (%):Organism Classification acid nucleotide Gene ID Pan paniscus chimpanzeeMammalia 98 98 XM_003810595 Mus musculus mouse Mammalia 87 75 NM_024281Bos mutus yak Mammalia 38 78 XM_005904566 Bos taurus cow Mammalia 87 74XM_003582930 Canis familiaris dog Mammalia 91 84 NM_001003179Monodelphis domestica oppossum Mammalia 83 68 XM_001382073 Pongo abeliiorangutan Mammalia 92 85 XM_003779288 Macaca mulatta monkey Mammalia 9185 XM_001086541 Macaca fascicularis monkey Mammalia 92 85 XM_005568285Cricetulus griseus Chinese hamster Mammalia 88 78 XM_003496471 Jaculusjaculus rodent Mammalia 86 76 XM_004668728 Otolemur garnettii galagoMammalia 90 79 XM_003801321 Spermophilus gopher Mammalia 88 78XM_005320449 tridecemlineatus Myotis brandtii bat Mammalia 88 79XM_005860586 Rattus norvegicus rat Mammalia 87 75 XM_230637 Mustelaputorius furo ferret Mammalia 88 78 XM_004754183 Microtus ochrogastervole Mammalia 87 74 XM_005365517 Octodon degus rodent Mammalia 85 74XM_004635679 Ochotona princeps pica Mammalia 87 74 XM_004585968Heterocephalus glaber rat Mammalia 86 75 XM_004909847 Callithrix jacchusmarmoset Mammalia 84 69 XM_002806762 Papio anubis baboon Mammalia 91 83XM_003905099 Gorilla gorilla gorilla gorilla Mammalia 93 88 XM_004061840Saimiri boliviensis monkey Mammalia 92 84 XM_003933177 boliviensisOdobenus rosmarus walrus Mammalia 87 76 XM_004412293 divergens Orcinusorca whale Mammalia 87 76 XM_004270397 Myotis lucifugus bat Mammalia 8876 XM_006087977 Equus caballus horse Mammalia 89 82 XM_005604466Ceratotherium rhinoceros Mammalia 90 81 XM_004433726 simum simum Dasypusnovemcinctus armadillo Mammalia 88 77 XM_004472392 Felis catus catMammalia 90 81 XM_003983802 Sus scrofa pig Mammalia 86 74 XM_005672713Trichechus manatus Trichechidae Mammalia 88 77 XM_004376447 latirostrisCondylura cristata mole Mammalia 89 78 XM_004687152 Pantholops hodgsoniiantelope Mammalia 87 77 XM_005985103 Chinchilla lanigera chinchillaMammalia 86 73 XM_005380908 Echinops telfairi tenrec Mammalia 88 75XM_004697814 Ovis aries sheep Mammalia 84 73 XM_004014691 Chrysemyspieta bellii turtle Reptilia 82 63 XM_005287452 Geospiza fortis birdAves 82 66 XM_005426202 Zonotrichia albicollis bird Aves 82 66XM_005488513 Pseudopodoces humilis bird Aves 81 64 XM_005523241Taeniopygia guttata Finch Aves 82 65 XM_002196328 Gallus gallusJunglefowl Aves 82 64 NM_001257346 Ficedula albicollis Flycatcher Aves81 65 XM_005042941 Alligator sinensis alligator Reptilia 81 62XM_006030436 Pelodiscus sinensis turtle Reptilia 80 61 XM_006118575Sarcophilus harrisii Tasmanian Mammalia 81 62 XM_003758168 devilMelopsittacus undulatus budgerigar Aves 81 63 XM_005152037 Latimeriachalumnae coelacanth Sarcopterygii 81 59 XM_005992924 Capra hircus goatMammalia 80 62 XM_005688109 Xenopus tropicalis frog Amphibia 78 55BC074706 Xenopus laevis frog Amphibia 78 55 NM_001089623 Mesocricetusauratus hamster Mammalia 80 57 XM_005068495 Danio rerio zebrafishActinopterygii 76 50 AY398408 Perca flavescens yellow perchActinopterygii 78 58 HQ206468

Thus, as referred to in the present invention, the “p180 protein” refersto:

(a) a protein that consists of an amino acid sequence with at least 70%sequence identity to the amino acid sequence (SEQ ID NO: 2) ofhuman-derived p180 protein, and which has a capability of promotingpolysome formation on an intracellular endoplasmic reticulum membrane;

(b) a protein that consists of an amino acid sequence derived from theamino acid sequence (SEQ ID NO: 2) of the human-derived p180 protein bydeletion, substitution, or addition of one or more amino acids, andwhich has a capability of promoting polysome formation on theintracellular endoplasmic reticulum membrane;

(c) a protein that consists of an amino acid sequence specified by anucleotide sequence with at least 70% sequence identity to thenucleotide sequence (SEQ ID NO: 1) of a gene encoding the human-derivedp180 protein, and which has a capability of promoting polysome formationon the intracellular endoplasmic reticulum membrane;

(d) a protein that consists of an amino acid sequence specified by anucleotide sequence derived from the nucleotide sequence (SEQ ID NO: 1)of the gene encoding the human-derived p180 protein by deletion,substitution, or addition of one or more nucleotides, and which has acapability of promoting polysome formation on the intracellularendoplasmic reticulum membrane; or

(e) a protein that consists of an amino acid sequence specified by anucleotide sequence hybridizable under stringent conditions with anucleotide sequence complementary to the nucleotide sequence (SEQ IDNO: 1) of the gene encoding the human-derived p180 protein, and whichhas a capability of promoting polysome formation on the intracellularendoplasmic reticulum membrane.

With regard to (a) in this mode, by the statement regarding amino acidsequence identity, which reads “with at least 70% sequence identity tothe amino acid sequence (SEQ ID NO: 2) of human-derived p180 protein”,it is meant that any value for percent sequence identity can be selectedfrom 70% to 100%, and examples of the percent sequence identity valuethat can be selected include 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

With regard to (b) in this mode, by “deletion, substitution, or additionof one or more amino acids”, it is meant that the number of amino acidsto be deleted, substituted or added ranges from 1 to about 10, andexamples of the number of amino acids that can be selected include 1, 2,3, 4, 5, 6, 7, 8, 9 and 10.

With regard to (c) in this mode, by “with at least 70% sequence identityto the nucleotide sequence (SEQ ID NO: 1) of a gene encoding thehuman-derived p180 protein”, it is meant that any value for percentsequence identity can be selected from 70% to 100%, and examples of thepercent sequence identity value that can be selected include 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,and 100%.

With regard to (d) in this mode, by “deletion, substitution, or additionof one or more nucleotides”, it is meant that the number of nucleotidesto be deleted, substituted or added ranges from 1 to about 10, andexamples of the number of nucleotides that can be selected include 1, 2,3, 4, 5, 6, 7, 8, 9 and 10. Additionally, it is presupposed that such“deletion, substitution, or addition of one or more nucleotides” makesit possible to specify a protein having a desired function, withoutintroduction of a stop codon.

With regard to (e) in this mode, by “under stringent conditions”, it ismeant that the hybridization conditions can be easily determined by aperson having an ordinary skill in the art on the basis of, for example,the nucleotide sequence length of a gene. As typically described inCurrent Protocols in Molecular Biology, vol. 1 (John Wiley and Sons,Inc.) and Molecular Cloning, 2nd Edition (Sambrook, et al. (1989)),exemplary basic hybridization conditions are hybridization in 5×SSC,5×Denhardt's solution, and 1% SDS at 25-68° C. for several hours toovernight. In this case, the hybridization temperature is preferably inthe range of 45-68° C. (no formamide) or 30-42° C. (50% formamide).Exemplary washing conditions are washing in 0.2×SSC at 45-68° C. It iswell known to those skilled in the art that determining hybridizationconditions including formamide concentration, salt concentration andtemperature as appropriate enables cloning of nucleic acid moleculescomprising a nucleotide sequence with a sequence identity above acertain level; and the thus cloned nucleic acid molecules are allincluded in the scope of the present invention.

The full length of the human p180 protein is the protein having theamino acid sequence of SEQ ID NO: 2 (GenBank Accession No. AB287347),and this protein is encoded by the nucleotide sequence of SEQ ID NO: 1(GenBank Accession No. AB287347). Also, the aforementioned murine p180protein is encoded by the nucleotide sequence of GenBank Accession No.NP_077243, the aforementioned rat p180 protein is encoded by thenucleotide sequence of GenBank Accession No. XP_230637, theaforementioned Chinese hamster p180 protein is encoded by the nucleotidesequence of GenBank Accession No. XM_003496471, the aforementionedcanine p180 protein is encoded by the nucleotide sequence of GenBankAccession No. NP_001003179, the aforementioned equine p180 protein isencoded by the nucleotide sequence of GenBank Accession No.XP_001915027, the aforementioned simian p180 protein is encoded by thenucleotide sequence of GenBank Accession No. XP_002798281, theaforementioned chimpanzee p180 protein is encoded by the nucleotidesequence of GenBank Accession No. XP_514527, and the aforementionedporcine p180 protein is encoded by the nucleotide sequence of GenBankAccession No. XP_001926148.

For example, when the p180 protein used is the human p180 protein,polysome formation on an intracellular endoplasmic reticulum can bepromoted by expressing a portion comprising any of the followingregions: a region consisting of the amino acids at positions 27 to 157of a protein having the amino acid sequence of SEQ ID NO: 2 (human p180protein); a region consisting of the amino acids at positions 623 to 737of said protein; a region consisting of the amino acids at positions 738to 944 of said protein; and a region consisting of the amino acids atpositions 945 to 1540 of said protein (Patent Literature 1).

Thus, as referred to in the present invention, the “portion of p180protein” refers to, for example: a portion comprising an amino acidsequence corresponding to a region consisting of the amino acids atpositions 27 to 157 of a protein having the amino acid sequence of SEQID NO: 2 (human p180 protein); a portion comprising an amino acidsequence corresponding to a region consisting of the amino acids atpositions 623 to 737 of said protein; a portion comprising an amino acidsequence corresponding to a region consisting of the amino acids atpositions 738 to 944 of said protein; or a portion comprising an aminoacid sequence corresponding to a region consisting of the amino acids atpositions 945 to 1540 of said protein. Proteins comprising such aportion can have a capability of promoting polysome formation. Withregard to humans, examples of the thus-specified portion of the p180protein include not only such portions as mentioned above, per se,including: a portion comprising a region consisting of the amino acidsat positions 27 to 157 of a protein having the amino acid sequence ofSEQ ID NO: 2 (human p180 protein); a portion comprising a regionconsisting of the amino acids at positions 623 to 737 of said protein; aportion comprising a region consisting of the amino acids at positions738 to 944 of said protein; and a portion comprising a region consistingof the amino acids at positions 945 to 1540 of said protein, but alsoMTB-2 domain adjacent to the C-terminal side of the N-terminaltransmembrane domain of the human p180 protein, or a highly basicN-terminal region containing a ribosome-binding repeat domain, a highlybasic tandem repeat domain, or a microtubule binding and bundling domain(MTB-1 domain) (Patent Literature 1).

As mentioned above, also in the cases of using portions of othermammalian p180 proteins, since the amino acid sequence of the human p180protein and the amino acid sequences of other mammalian p180 proteinsare generally highly conserved, the amino acid sequences of fragmentscomprising portions or regions corresponding to the amino acid sequencesof the proteins (a) to (e) mentioned above can also be used as the“portion of the p180 protein”.

The aforementioned recombinant cell of the present invention is secondlycharacterized by having enhanced intracellular expression of a proteinpromoting mRNA localization to an endoplasmic reticulum (ER). Examplesof such a protein promoting mRNA localization to an endoplasmicreticulum (ER) include SF3b4 protein, especially the full length or aportion of a mammalian SF3b4 protein (e.g., the full-length amino acidsequence 424 AA of SEQ ID NO: 4; RRM1 consisting of 13 to 91 AA of SEQID NO: 4; RRM2 consisting of 100 to 179 AA of SEQ ID NO: 4; a C-terminalregion consisting of 180 to 424 AA of SEQ ID NO: 4).

In particular, the SF3b4 protein is a protein that is generally detectedonly in the nucleus. However, further detailed investigation found thatin fibroblasts actively secreting collagen, most of the SF3b4 protein isdetected in the nucleus but some of said protein is found in themembrane fraction containing cytoplasmic endoplasmic reticulum. TheSF3b4 protein (also referred to as “SAP49/SF3b49”) is a substance thatis classified as an RNA recognition motif (RRM)-type RNA-binding protein(RBP) family due to containing two RNA recognition motifs (RRM) on itsamino-terminal side, and which has a proline-rich domain with an unknownfunction on its carboxy-terminal side. The normal splicing reactionprocess requires both of these two RNA recognition motifs (RRM) (RRM1,RRM2). These motifs are highly conserved even in yeast, and are presumedto constitute an important functional domain. Further, the SF3b4 proteinbinds to the other constitutional protein of an SF3b complex, i.e.,SAP145 protein, and this binding is also shown to require both of thetwo RNA recognition motifs (RRM) (RRM1, RRM2) (Champion-Arnaud & Reed,1994). Thus, it is considered that the RRM domain of the SF3b4 proteinnot only requires RNA recognition but also acts on a protein-proteininteraction.

In this connection, speaking of the comparison between the amino acidsequences of SF3b4 proteins, it is known that the SF3b4 proteins of allthe mammalian species investigated show 100% amino acid sequencesimilarity to the human SF3b4 protein, and that also in the case ofother species, the yeast and insect SF3b4 proteins have amino acidsequence similarities of 40 to 54% and 63 to 81%, respectively, to thehuman SF3b4 protein—thus, the SF3b4 protein is reported to be a veryconservative protein in all living organisms.

TABLE 2 Sequence homologies of SF3b4 of various species to human SF3b4Protein Gene similarity identity to human to human (%): (%): OrganismClassification amino acid nucleotide Gene ID Pan troglodytes chimpanzeeMammalia 100 99 XM_513768 Gorilla gorilla gorilla gorilla Mammalia 10099 XM_004026557 Papio anubis baboon Mammalia 100 97 XM_003892563 Panpaniscus bonobo Mammalia 100 97 XM_003817322 Callithrix jacchus marmosetMammalia 100 98 XM_002759857 Mus musculus mouse Mammalia 100 92NM_153053 Bos mutus yak Mammalia 100 95 XM_005894874 Bos taurus cowMammalia 100 95 NM_001205584 Tursiops truncatus dolphin Mammalia 100 96XM_004315506 Macaca mulatta monkey Mammalia 100 99 NM_001261232 Macacafascicularis monkey Mammalia 100 97 XM_005542035 Canis lupus wolfMammalia 100 93 XM_540295 Cricetulus griseus Chinese hamster Mammalia100 91 XM_003498632 Jaculus jaculus rodent Mammalia 100 88 XM_004668225Otolemur garnettii galago Mammalia 100 95 XM_003800416 Spermophilustridecemlineatus gopher Mammalia 100 94 XM_005331144 Rattus norvegicusrat Mammalia 100 91 NM_001011951 Mustela putorius furo ferret Mammalia100 94 XM_004776825 Microtus ochrogaster vole Mammalia 100 91XM_005356945 Ochotona princeps pica Mammalia 100 91 XM_004588838Heterocephalus glaber rat Mammalia 100 94 XM_004853946 Saimiriboliviensis boliviensis monkey Mammalia 100 96 XM_003941980 Odobenusrosmarus divergens walrus Mammalia 100 94 XM_004404182 Orcinus orcawhale Mammalia 100 95 XM_004285129 Myotis lucifugus bat Mammalia 100 94XM_006095177 Equus caballus horse Mammalia 100 95 XM_001488599 Caprahircus goat Mammalia 100 95 XM_005677701 Mesocricetus auratus Goldenhamster Mammalia 100 92 XM_005084248 Sarcophilus harrisii Tasmaniandevil Mammalia 100 86 XM_003769946 Cavia porcellus guinea pig Mammalia100 93 XM_003478880 Sorex araneus shrew Mammalia 100 92 XM_004618056Ceratotherium simum simum rhinoceros Mammalia 100 95 XM_004435941Dasypus novemcinctus armadillo Mammalia 100 91 XM_004461651 Felis catuscat Mammalia 100 95 XM_004001496 Sus scrota pig Mammalia 100 95XM_001926489 Trichechus manatus latirostris Trichechidae Mammalia 100 94XM_004389600 Condylura cristata mole Mammalia 100 92 XM_004689571Pantholops hodgsonii antelope Mammalia 100 95 XM_005970739 Chinchillalanigera chinchilla Mammalia 100 93 XM_005378708 Echinops telfairitenrec Mammalia 100 93 XM_004716571 Ovis aries sheep Mammalia 100 95XM_004002436 Geospiza fortis bird Aves 98 77 XM_005430326 Pseudopodoceshumilis bird Aves 98 84 XM_005533471 Gallus gallus Junglefowl Aves 98 81XM_423721 Falco peregrinus Peregrine Falcon Aves 97 74 XM_005244315Chrysemys picta bellii turtle Reptilia 97 83 XM_005293645 Alligatorsinensis alligator Reptilia 96 79 XM_006033517 Danio rerio zebrafishActinopterygii 92 79 NM_153661 Pundamilia nyererei lapsus Actinopterygii91 80 XM_005728504 Maylandia zebra Mbuna Actinopterygii 91 80XM_004549154 Capsaspora owczarzaki monotypic genus Filasterea 88 77XM_004345599 Xenopus tropicalis frog Amphibia 88 79 NM_203785 Xenopuslaevis frog Amphibia 87 78 NM_001086631 Aplysia californica Californiasea hare Gastropoda 77 66 XM_005109109 Drosophila melanogaster fruit flyInsecta 81 63 NM_078503.41 Anopheles gambiae mosquito Insecta 75 65XM_321584.4 Apis mellifera honey bee Insecta 63 57 Group13.4(31702-33391) Caenorhabditis elegans worm Secernentea 73 65 II(8034428-8035787)Daphnia pulex common water flea Crustacea 79 65 scaffold_1(3898136-3899124) Trichoplax adhaerens Trichoplax Tricoplacia 76 64scaffold_6(4430698- 4432902) Nematostella vectensis sea anemone Anthozoa68 62 scaffold_59(938974- 944852) Strongylocentrotus purpuratus seaurchin Echinoidea 66 62 scaffold_1412 Schistosoma mansoni schistosomeparasite Trematoda 65 60 Smp_scaff000217(455 137-459876) Arabidopsisthaliana thale cress eudicotyledons 65 60 NM_127407.3 Phytophthorainfestans Chromalveolata Oomycota 62 56 Supercont1.42(913888- 914494)Neurospora crassa bread mold Ascomycetes 52 53 XM_956049.1Schizosaccharomyces pombe fission yeast Schizosaccharomycetes 54 55NM_001019427 Saccharomyces cerevisiae baker's yeast Saccharomycetes 4150 NP_014964 Ashbya gossypii A. gossypii yeast Saccharomycetes 41 50NM_208241.1 Kluyveromyces lactis K. lactis yeast Saccharomycetes 40 47XM_452844.1 Chlamydomonas reinhardtii green algae Chlorophyceae 50 46DS496110 Chlamydomonas reinhardtii green alga Chlorophyceae 50 56XM_001696328

Given the aforementioned fact that the primary amino acid sequence ofthe SF3b4 protein is widely and highly conserved beyond biologicalspecies, it is easily presumed that a function verified using the humanSF3b4 protein would be reproduced also in the case of using SF3b4proteins derived from other biological species.

Thus, as referred to in the present invention, the “SF3b4 protein”refers to:

(i) a protein that consists of an amino acid sequence with at least 70%sequence identity to the amino acid sequence (SEQ ID NO: 4) ofhuman-derived SF3b4 protein, and which has a capability of promotingmRNA localization to an endoplasmic reticulum;

(ii) a protein that consists of an amino acid sequence derived from theamino acid sequence (SEQ ID NO: 4) of the human-derived SF3b4 protein bydeletion, substitution, or addition of one or more amino acids, andwhich has a capability of promoting mRNA localization to the endoplasmicreticulum;

(iii) a protein that consists of an amino acid sequence specified by anucleotide sequence with at least 70% sequence identity to thenucleotide sequence (SEQ ID NO: 3) of a gene encoding the human-derivedSF3b4 protein, and which has a capability of promoting mRNA localizationto the endoplasmic reticulum;

(iv) a protein that consists of an amino acid sequence specified by anucleotide sequence derived from the nucleotide sequence (SEQ ID NO: 3)of the gene encoding the human-derived SF3b4 protein by deletion,substitution, or addition of one or more nucleotides, and which has acapability of promoting mRNA localization to the endoplasmic reticulum;or

(v) a protein that consists of an amino acid sequence specified by anucleotide sequence hybridizable under stringent conditions with anucleotide sequence complementary to the nucleotide sequence (SEQ ID NO:3) of the gene encoding the human-derived SF3b4 protein, and which has acapability of promoting mRNA localization to the endoplasmic reticulum.

With regard to (i) in this mode, by the statement regarding amino acidsequence identity, which reads “with at least 70% sequence identity tothe amino acid sequence (SEQ ID NO: 4) of human-derived SF3b4 protein”,it is meant that any value for percent sequence identity can be selectedfrom 70% to 100%, and examples of the percent sequence identity valuethat can be selected include 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

With regard to (ii) in this mode, by “deletion, substitution, oraddition of one or more amino acids”, it is meant that the number ofamino acids to be deleted, substituted or added ranges from 1 to about10, and examples of the number of amino acids that can be selectedinclude 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

With regard to (iii) in this mode, by “with at least 70% sequenceidentity to the nucleotide sequence (SEQ ID NO: 3) of a gene encodingthe human-derived SF3b4 protein”, it is meant that any value for percentsequence identity can be selected from 70% to 100%, and examples of thepercent sequence identity value that can be selected include 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,and 100%.

With regard to (iv) in this mode, by “deletion, substitution, oraddition of one or more nucleotides”, it is meant that the number ofnucleotides to be deleted, substituted or added ranges from 1 to about10, and examples of the number of nucleotides that can be selectedinclude 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. Additionally, it ispresupposed that such “deletion, substitution, or addition of one ormore nucleotides” makes it possible to specify a protein having adesired function, without introduction of a stop codon.

With regard to (v) in this mode, by “under stringent conditions”, it ismeant that the hybridization conditions that can be adopted are asdescribed above in relation to the p180 protein.

The full length of the human SF3b4 protein is the protein having theamino acid sequence of SEQ ID NO: 4 (GenBank Accession No. NP_005841.1),and this protein is encoded by the nucleotide sequence of SEQ ID NO: 3(GenBank Accession No. NP_005841.1). Also, the aforementioned murineSF3b4 protein is encoded by the nucleotide sequence of GenBank AccessionNo. NP_694693.1, the aforementioned rat SF3b4 protein is encoded by thenucleotide sequence of GenBank Accession No. NP_001011951.1, theaforementioned Chinese hamster SF3b4 protein is encoded by thenucleotide sequence of GenBank Accession No. XP_003498680.1, theaforementioned canine SF3b4 protein is encoded by the nucleotidesequence of GenBank Accession No. XP_540295.3, the aforementioned equineSF3b4 protein is encoded by the nucleotide sequence of GenBank AccessionNo. XP_001488649.2, the aforementioned simian SF3b4 protein is encodedby the nucleotide sequence of GenBank Accession No. NP_001097793.1, theaforementioned chimpanzee SF3b4 protein is encoded by the nucleotidesequence of GenBank Accession No. XP_513768.2, and the aforementionedporcine SF3b4 protein is encoded by the nucleotide sequence of GenBankAccession No. XP_001926524.1.

When a DNA encoding a protein as a product of interest which is requiredto be expressed in the present invention is transformed into the cell tobe used in a protein expression system, the mRNA (precursor) transcribedfrom the DNA is converted into a mature mRNA through removal of intronswith no genetic information on amino acids by splicing. This process isassumed by a spliceosome, a small nuclear RNA (snRNA)-proteinmacrocomplex. The spliceosome has five types of low-molecular-weightribonucleoprotein complexes (snRNPs), and among these snRNPs, the SF3b4protein is a constituent of U2-snRNP, which contains an RNA-bindingdomain.

There had hitherto been no report that splicing factors including SF3b4protein perform some function at the protein translation level. However,the analysis made by the present inventors found that levels of SF3b4protein in the membrane fraction containing endoplasmic reticulumsignificantly increases, which occurs concomitantly with the SF3b4protein association with mRNA and binding to the coiled-coil domain ofp180 protein, thereby promoting mRNA localization to the endoplasmicreticulum, and consequently the secretory capacity in the cultured cellscan be enhanced.

In other words, it was found that, when a nucleic acid molecule encodinga protein as a product of interest is transformed in the recombinantcell having enhanced expression of either or both of these two types ofproteins, the mRNA transcribed from the DNA encoding the protein as aproduct of interest acts with the SF3b4 protein expressedintracellularly or interacts with the p180 protein expressedintracellularly, or the mRNA transcribed from the DNA encoding theprotein as a product of interest interacts with the SF3b4 protein andthen the coiled-coil domain of the p180 protein interacts with the SF3b4protein, whereupon mRNA localization to an endoplasmic reticulum ispromoted thereby enhancing the cell's synthetic or secretory capacity ofthe protein as a product of interest.

All types of fibrosis which can cause long-term injury to varioustissues to fibrose them are unknown as to their cause, detailedmechanism of development, and effective therapy, and are poor inprognosis. For example, in the case of idiopathic pulmonary fibrosis,the fibrosis is believed to progress because of an increase in theproduction of collagen and the like to repair alveolar epithelium injuryresulting from various stimuli, which leads to an abnormal repairreaction, but no effective therapy has been established. There had notbeen a clue to the prevention of abnormal increase in collagenproduction under such pathological conditions, but it was newly found inthe present invention that the SF3b4 protein, which was hithertobelieved to function as a splicing factor, plays an essential role incollagen synthesis/secretion, which indicated that collagen synthesiscan be suppressed by functional inhibition or expression suppression ofSF3b4. Since it is believed that the expression suppression of SF3b4 canbe achieved by administration of its specific shRNA or the like and thatthe functional inhibition of SF3b4 can be induced by various agentsinhibiting a splicing process, a possibility was suggested that thefunctional inhibition or expression suppression of SF3b4 may suppress anabnormal accumulation of collagen in fibrosis and prevent aggravation offibrosis.

The cell that can be used to prepare a recombinant cell in the presentinvention can be of any type as long as it is suitable for proteinexpression, and examples of the source cell that can be used includemammal-derived cells such as CHO cell, HEK293 cell and HeLa cell. Bytransfecting the aforementioned full length or portion p180 proteinand/or the aforementioned full length or portion of SF3b4 protein intothese cells using a method commonly used in the art, the full length orportion of p180 protein and/or the full length or portion SF3b4 proteincan be expressed in said cells.

In order to express the aforementioned full length or portion of p180protein and/or the full length or portion of SF3b4 protein in thesecells, a transformation method commonly used in the art can be used. Forthe purpose of the transformation, the following method can be used: aDNA encoding the full length or portion of p180 protein and/or a DNAencoding the full length or portion of SF3b4 protein are/is incorporatedinto an expression vector such as pcDNA, pEGFP or pCAGGS, and eachexpression vector is transformed into the cells.

In the present invention, the CHO cell-derived cell line CHO 5 g wasprepared as a recombinant cell stably expressing p180 protein, the CHOcell-derived cell line CHO 3D5 was prepared as a recombinant cell stablyexpressing SF3b4 protein; and the CHO cell-derived cell line CHO YA7 wasprepared as a recombinant cell having enhanced expression of these twoproteins at the same time (refer to Example 1 described below), andthese cell lines were deposited to the Patent Microorganisms Depositaryof the National Institute of Technology and Evaluation (the accessionnumber for the CHO 3D5 is NITE BP-01753; the accession number for theCHO YA7 cell is NITE BP-1535; or the accession number for the CHO 1B2cell is NITE ABP-01811).

The present inventors also demonstrated that there can be provided amethod in which, in a recombinant cell having enhanced expression of thefull length or a portion of p180 protein and/or the full length or aportion of SF3b4 protein, a nucleic acid molecule encoding the proteinas a product of interest is transformed or production of the protein asa product of interest is increased, whereby a synthetic or secretorycapacity of a protein as a product of interest is enhanced andconsequently the protein as a product of interest is produced.

In this method, the protein as a product of interest which is producedas a result of enhancing a synthetic or secretory capacity can be anyprotein intended to be produced by a biotechnological technique. Forexample, the protein as a product of interest can be exemplified byglycoproteins, and examples of the glycoproteins that can be selectedinclude, but are not limited to, antibodies, collagens, fibronectins,and laminins.

The present inventors provide, in the second mode of the presentinvention, a method for increasing an amount of a protein as a productof interest to be expressed in a cell as an expression system, wherein,in an expression unit for expressing the protein as a product ofinterest, a cic-element is inserted, downstream of a promoter andupstream of the start codon in the nucleotide sequence of a DNA encodingthe protein as a product of interest, whereby the amount of the proteinas a product of interest to be expressed in the cell as an expressionsystem is increased. The aforementioned insertion of the sequence of acis-element into an expression unit can be exemplified not only byinserting the sequence of a cis-element downstream of a promoter andupstream of the start codon in the nucleotide sequence of a DNA encodingthe protein as a product of interest, in an expression plasmid for theprotein as a product of interest, but also by inserting the sequence ofa cis-element downstream of a promoter and upstream of the ORF startcodon of the gene of interest, in a site-specific manner in a case wherethe promoter and the product of interest have already been geneticallyintroduced into the cell.

As described in the Background Art section, it has been shown that themRNAs for some proteins, including collagen genes, have a tendency toform a polysome in which multiple ribosomes, machines for proteinsynthesis, are attached to each other (Patent Literature 1, Non-patentLiterature 2). However, there have often been problems where only asmall amount of a protein as a product of interest was synthesized orsecreted in spite of the fact that a gene transcript encoding theprotein as a product of interest was expressed at a high level in cellstransfected with a DNA encoding the protein as a product of interest.This was presumed to be possibly because mRNA was not provided in anadequate manner to be used to machines for translation on theendoplasmic reticulum membranes in used cells.

The analysis made based on these considerations found that a cic-elementpresent in the 5′ untranslated region of a collagen gene has acapability of increasing an expressed protein amount. More specifically,it was considered that an RRM protein recognizing the cis-elementsequence in the 5′ untranslated region of a mature mRNA binds to saidsequence, thereby leading to an augmentation of mRNAtransport/localization onto the endoplasmic reticulum membrane, a placeof synthesis of secretory proteins, and to a further increase intranslation efficiency.

The cis-element observed in the present invention was proved from theresults of the analysis of a type I collagen gene, and the nucleotidesequence of this cis-element was found to be present in the 5′untranslated region of a type I collagen gene. Therefore, in the presentinvention, the nucleotide sequence of such a cis-element can beexemplified by a sequence derived from the nucleotide sequence of the 5′untranslated region of a type I collagen gene, but cis-element sequencesderived from other genes, such as those listed below, can also be usedas long as they have a desired effect: a sequence derived from thenucleotide sequence of the 5′ untranslated region of a fibronectin gene;a sequence derived from the nucleotide sequence of the 5′ untranslatedregion of the matrix metalloproteinase 14 (MMP14) gene; a sequencederived from the nucleotide sequence of the 5′ untranslated region ofthe prolyl 4-hydroxylase A2 (P4HA2) gene; and a sequence derived fromthe nucleotide sequence of the 5′ untranslated region of the prolyl4-hydroxylase A1 (P4HA1) gene.

The cis-element that can be used in the present invention isstructurally characterized by containing one or more 9- to 12-nucleotidemotifs “GAN₁-(X)_(n)-ACN₂” (n=3 to 6) (N₁ and N₂ can be independentlyany of the nucleotides A, T, C and G) in the 5′ untranslated region of agene present in an expression plasmid for expressing a protein as aproduct of interest. Specific examples of the motifs include motifspresent as native cis-elements, which are characterized in that N₁ is Gand that N₂ is A or G or C. More specifically, such motifs can beexpressed as “GAG xxx ACV” (SEQ ID NO: 17), “GAG xxxx ACV” (SEQ ID NO:18), “GAG xxxxx ACV” (SEQ ID NO: 19), and “GAG xxxxxx ACV” (SEQ ID NO:20) (in these sequences, V represents A or G or C). For example, it wasfound that in the case of a type I collagen gene, four motifs areincluded in the 5′ untranslated region.

In the case of using a cis-element derived from a type I collagen gene,the nucleotide sequence of the cis-element that can be used is anysequence selected from the group consisting of the full length of SEQ IDNO: 5 or the full length of SEQ ID NO: 7, and the nucleotides atpositions 1 to 102, positions 1 to 78, positions 1 to 60, positions 61to 126, positions 16 to 57, positions 79 to 126, positions 103 to 126,positions 58 to 78, positions 51 to 78, positions 1 to 27, and positions70 to 78 of SEQ ID NO: 5.

Also, in the case of using a cis-element derived from a fibronectingene, the nucleotide sequence of the cis-element that can be used is anysequence selected from the group consisting of the full length of SEQ IDNO: 6 and the full length of SEQ ID NO: 8.

TABLE 3 Table 3: List of the sequences of cis-elements Cis- SEQ ele- IDment Sequence NO #1 tcgtcggagc agacgggagt ttctcctcgg ggtcggagca 5ggaggcacgc ggagtgtgag gccacgcatg agcggacgctaaccccctcc ccagccacaa agagtctaca tgtctagggt ctagac #2gcccgcgccg gctgtgctgc acagggggag gagagggaac 6cccaggcgcg agcgggaaga ggggacctgc agccacaacttctctggtcc tctgcatccc ttctgtccct ccacccgtccccttccccac cctctggccc ccaccttctt ggaggcgacaacccccggga ggcattagaa gggatttttc ccgcaggttgcgaagggaag caaacttggt ggcaacttgc ctcccggtgc gggcgtctct cccccaccgt ctcaac#3 tcgtcggagc agacgggagt ttctcct 7 #4ccaccttctt ggaggcgaca acccccggga gg 8 #5tcgtcggagc agacgggagt ttctcctcgg ggtcggagca 21 ggaggcacgc ggagtgtgag #6gccacgcatg agcggacgct aaccccctcc ccagccacaa 22agagtctaca tgtctagggt ctagac #7aacgggcgcc gcggcgggga gaagacgcag agcgctgctg 23ggctgccggg tctcccgctt ccccctcctg ctccaagggcctcctgcatg agggcgcggt agagacccgg acccgcgccgtgctcctgcc gtttcgctgc gctccgcccg ggcccggctc agccaggccc cgcggtgagc c #8cagggggagg agagggaacc ccaggcgcga 24 #9gagcgggaag aggggacctg cagccacaac tt 25 #10cagggggagg agagggaacc ccaggcgcga gcgggaagag 26 gggacctgca gccacaactt #11tcgtcggagc agacgggagt ttctcctcgg ggtcggagca 27ggaggcacgc ggagtgtgag gccacgcatg agcggacgctaaccccctcc ccagccacaa agagtctaca tgt

Expression plasmids containing such a cis-element can be used not onlyin intact host cells, but also in the cells prepared in the presentinvention: i.e., a cell having enhanced expression of the full length ora portion of p180 protein, a cell having enhanced expression of the fulllength or a portion of splicing factor 3B subunit 4 (SF3b4) protein, ora cell having enhanced expression of both of these proteins.

Examples are provided herein below for the purpose of more specificallydescribing the present invention described above, but the examplesprovided below are not intended to limit this invention.

EXAMPLES Example 1: Establishment of a Cell Line Expressing SF3b4Protein, or Coexpressing p180 Protein and SF3b4 Protein

Preparation of Plasmids

Establishment of a cell line expressing SF3b4 protein, or coexpressingp180 protein and SF3b4 protein was achieved by separately preparing anexpression plasmid containing a nucleic acid encoding p180 protein andan expression plasmid containing a nucleic acid encoding SF3b4 protein,and sequentially transfecting these plasmids into CHO cells.

The expression plasmid encoding the full length of human p180 protein(GenBank Accession No. AB287347), pcDNA-p180/54R, was prepared accordingto the procedure described in Patent Literature 1 (JP 2005-312409).

The expression plasmid encoding the full length of human SF3b4 protein,pEF-SF3b4, was prepared according to the procedure described below. Morespecifically, the cDNA sequence encoding the full length of human SF3b4protein (GenBank Accession No. NP_005841.1) was amplified by PCR, andthen inserted and ligated into the KpnI-EcoRV site of pEF1/Myc-Hisvector (produced by Life Technologies) to thereby obtain the plasmidpEF-SF3b4.

The expression plasmid encoding the full length of Chinese hamster SF3b4protein, pEF-CHO-SF3b4, was prepared according to the proceduredescribed below. More specifically, total RNA was extracted from CHOcells, and the cDNA sequence encoding the full length of Chinese hamsterSF3b4 protein (GenBank Accession No. XP_003498680.1) was amplified byPCR. Then, the cDNA sequence was inserted and ligated into theKpnI-EcoRV site of pEF1/Myc-His vector to thereby obtain the plasmidpEF-CHO-SF3b4.

The expression plasmid encoding the sequence of a human cis-element(e.g., cis-elements #1 to #11), pEF-Cis, was prepared according to theprocedure described below. More specifically, a nucleic acid sequenceencoding a human cis-element (e.g., SEQ ID NO: 5 in the case ofcis-element #1) was amplified by PCR, and then inserted and ligated intothe BglII-HindIII site of pEGFP vector (produced by Clontech) to therebyobtain the plasmid prCMV-cis#-SEAP. The details of expression vectorseach containing a different cis-element will be described in Example 9.

Preparation of Cells Stably Expressing p180 Protein

Establishment of CHO cells stably expressing human p180 protein wasachieved according to the procedure described in Patent Literature 1.More specifically, the human p180 protein-expressing plasmidpcDNA-p180/54R was transfected into CHO cells by a lipofection method,and then the transfected cells were cultured in the presence of 400μg/mL of zeocin, whereby drug selection was done. After the culture for10 days, zeocin-resistant cell line colonies were isolated to establishthe cell line CHO 5 g which stably expresses p180 protein.

Preparation of Cells Stably Expressing SF3b4 Protein

To prepare CHO cells stably expressing human SF3b4 protein, the humanSF3b4 protein-expressing plasmid pEF-SF3b4 was transfected into CHOcells by a lipofection method, and then the transfected cells werecultured in the presence of 400 μg/mL of G418, whereby drug selectionwas done. After the culture for 10 days, G418-resistant cell linecolonies were isolated to establish the cell line CHO 3D5 which stablyexpresses SF3b4 protein (deposited to the Patent MicroorganismsDepositary of the National Institute of Technology and Evaluation(NPMD), 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan; AccessionNumber: NITE BP-01753; Deposit date: Nov. 21, 2013).

Preparation of Cells Stably Coexpressing p180 Protein and SF3b4 Protein

Next, to establish CHO cells stably coexpressing human p180 protein andhuman SF3b4 protein, the SF3b4 protein-expressing plasmid pEF-SF3b4 wastransfected into the cell line CHO 5 g by a lipofection method. Then,the transfected cells were cultured in the presence of 400 μg/mL of G418and 100 μg/mL of zeocin, whereby drug selection was done. After theculture for 14 days, G418- and zeocin-resistant cell line colonies wereisolated to establish the cell line CHO YA7 which stably coexpresseshuman p180 protein and human SF3b4 protein (deposited to the PatentMicroorganisms Depositary of the National Institute of Technology andEvaluation (NPMD), 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan;Accession Number: NITE BP-1535; Deposit date: Feb. 13, 2013).

Thereafter, to establish CHO cells stably coexpressing human p180protein and Chinese hamster SF3b4 protein, a plasmid expressing humanp180 and Chinese hamster SF3b4 was transfected into CHO cells by alipofection method. Then, the transfected cells were cultured in thepresence of 300 μg/mL of hygromycin, whereby drug selection was done.After the culture for 14 days, hygromycin-resistant cell line colonieswere isolated to establish the cell line CHO 1B2 which stably expresseshuman p180 protein and Chinese hamster SF3b4 protein (deposited to thePatent Microorganisms Depositary of the National Institute of Technologyand Evaluation (NPMD), 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan;Accession Number: NITE ABP-01811; Deposit date: Mar. 4, 2014).

Verification of Cell Properties

To verify that CHO 5 g cells stably expressed p180 protein, that CHO 3D5cells stably expressed SF3b4 protein, and that CHO YA7 cells and CHO 1B2cells coexpressed p180 protein and SF3b4 protein, CHO 5 g cells, CHO 3D5cells, CHO YA7 cells, and CHO 1B2 cells were each cultured in aDulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetalbovine serum at 37° C. in the presence of 5% CO₂. CHO cells were alsocultured as control cells.

After 40 hours, 1×10⁵ of each type of the cells suspended by trypsintreatment were harvested by centrifugation as samples for analysis, andthen analyzed by Western blotting for intracellularly expressed p180protein using an anti-p180 antibody (refer to Ogawa-Goto, K. et. al., J.Virol., 76 (2002) 2350-2362), and for intracellularly expressed SF3b4protein using an anti-SF3b4 antibody (produced by Santacruz).

According to FIG. 1, as for the CHO cells, the expressed p180 amount wasbelow the limit of detection, and SF3b4 protein was observed to beendogenously expressed at a low level (lane 1). As for the CHO 3D5cells, the expressed p180 protein amount was below the limit ofdetection as in the case of the CHO cells, but SF3B4 protein was highlyexpressed (lane 2). As for the CHO 5 g cells, p180 protein was highlyexpressed, but SF3b4 protein was observed to be endogenously expressedat a low level (lane 3). As for the CHO YA7 cells and the CHO 1B2 cells,p180 protein and SF3b4 protein were both highly expressed (lanes 4 and6). These results verified that there were successfully established theCHO 5 g cells highly expressing p180 protein, the CHO 3D5 cells highlyexpressing SF3b4 protein, and the CHO cell-derived cell lines CHO YA7and CHO 1B2 coexpressing p180 protein and SF3b4 protein.

Example 2: Activation of Secretion by p180 Protein Expression and/orSF3b4 Protein Expression

Using the cell lines prepared in Example 1, i.e., the CHO 5 g cellsexpressing p180 protein, the CHO 3D5 cells expressing SF3b4 protein, andthe cell line CHO YA7 coexpressing p180 protein and SF3b4 protein,investigation was made of p180 protein expression and/or SF3b4 proteinexpression, and activation of protein secretion.

An expression plasmid for human placental secreted alkaline phosphatase(SEAP) as a secretory marker was constructed according to the proceduredescribed below. More specifically, a cDNA fragment encoding the fulllength of SEAP protein (GenBank Accession No. NP_001623.3) was insertedand ligated into the NheI-XhoI site of an expression vector formammalian cells (pEGFP-C3; produced by Clontech) to thereby obtain theSEAP protein expression plasmid prCMV-SEAP.

To evaluate the respective types of cells for the secretory capacity ofSEAP protein, the SEAP expression plasmid and aβ-galactosidase-expressing plasmid for internal normalization, pEF1-LacZ(produced by Life Technologies), were cotransfected into each of the CHO3D5 cells, the CHO 5 g cells, the CHO YA7 cells, and the CHO cells usingthe Lipofectamine LTX reagent (produced by Life Technologies). Thetransfected cells were cultured in a DMEM supplemented with 0.1% fetalbovine serum for 20 hours, and then the culture supernatant was mixedwith a substrate solution containing p-Nitrophenyl phosphate (pNPP;produced by Sigma). After the reaction at room temperature for 30minutes, the mixture was measured for absorbance at a wavelength of 405nm using an absorbance spectrophotometer. The β-galactosidase activityin cell fractions was measured according to the standard protocol forthe β-Galactosidase Enzyme Assay System (produced by Promega).

The SEAP activities normalized by the measured β-galactosidase valuesare shown in FIG. 2. In the cases of the CHO 5 g cells expressing p180protein alone and the CHO 3D5 cells expressing SF3b4 protein alone, theSEAP secretory activity in culture supernatant increased significantlyby 1.7 to 2.0 times as compared to the CHO cells (FIG. 2). In the caseof the CHO YA7 cells coexpressing p180 protein and SF3b4 protein, theSEAP activity increased more significantly by 3.1 times as compared tothe CHO cells. These facts demonstrated that in both cases of theexpression of p180 protein alone and the expression of SF3b4 proteinalone, the SEAP secretory activity was increased significantly ascompared to the case of the CHO cells, but in the case of thecoexpression of p180 protein and SF3b4 protein, the secretory capacityin cells was increased more remarkably than in the case of the CHOcells.

Also, as compared to the SEAP activity of the CHO cells, which was takenas 1, the SEAP activity ratio of the CHO 1B2 cells was 3.1, whichindicates that the CHO 1B2 cells showed a remarkable increase in SEAPactivity. This fact demonstrated that Chinese hamster SF3b4 highlysimilar to human SF3b4 has comparable secretion enhancement activity tohuman SF3b4.

Example 3: Promotion of mRNA Localization to a Membrane Fraction byCoexpression of p180 Protein and SF3b4 Protein

Using the cell lines prepared in Example 1, i.e., the cell line CHO 5 gexpressing p180 protein, the cell line CHO 3D5 expressing SF3b4 protein,and the cell line CHO YA7 coexpressing p180 protein and SF3b4 protein,investigation was made of the relationship of p180 protein expressionand/or SF3b4 protein expression with promotion of mRNA localization to amembrane fraction.

The aforementioned SEAP expression plasmid was transfected into each ofthe CHO YA7 cells and the control cells using the Lipofectamine LTXreagent, and after 40 hours, the transfected cells were fractionatedinto a cytoplasmic fraction and a membrane fraction. The fractionationwas performed according to the procedure described in Non-patentLiterature 1 (Ueno, et al., (2010) J Biol Chem 285, 29941-29950). RNAwas extracted from each of these fractions according to the standardprotocol for the Trizol-LS reagent (produced by Life Technologies).Then, quantitative PCR was performed with SEAP-specific primers toquantitate SEAP mRNA (FIG. 3).

The results showed that the total mRNA content in both of thecytoplasmic and membrane fractions was almost the same among the CHO 3D5cells, the CHO 5 g cells, the CI-TO YA7 cells, and the CI-TO cells.However, about 30% of the total mRNA was present in the membranefraction in the cases of the CHO cells, the CHO 3D5 cells, and the CHO 5g cells, whereas in the case of the CHO YA7 cells, about 70% of thetotal mRNA was localized in the membrane fraction—mRNA localization wasgreatly shifted from the cytoplasm to the membrane fraction.

These facts demonstrated that the CHO YA7 cells coexpressing p180protein and SF3b4 protein have a capability of actively localizing mRNAto a membrane fraction during biosynthesis of secretory proteins.

Example 4: Construction of an Expression Vector in which a Cis-Elementis Inserted into a Secreted Alkaline Phosphatase Expression Unit

In this example, using the cell lines prepared in Example 1, i.e., thecell line CHO 3D5 expressing SF3b4 protein, and the cell line CHO YA7coexpressing p180 protein and SF3b4 protein, investigation was made ofthe structure of an expression plasmid for the purpose of furtherincreasing protein expression efficiency.

An expression vector in which a cis-element was inserted into the SEAPexpression plasmid prCMV-SEAP mentioned in Example 2 was constructedaccording to the procedure described below. RNA derived from humanfibroblasts was prepared according to the procedure described inNon-patent Literature 1 (Ueno, et al., (2010) J Biol Chem 285,29941-29950), and RT-PCR was performed using the prepared RNA as atemplate. In the process of amplification, cis-element #1 (SEQ ID NO: 5)derived from human type I collagen α1 was amplified using the primers(SEQ ID NO: 15, SEQ ID NO: 16) in which the BglII and HindIIIrecognition sequences were added towards the 5′ and 3′ ends,respectively. The amplified fragment was treated with BglII-HindIII, andthen inserted and ligated into the BglII-HindIII site located betweenthe CMV promoter and SEAP ORF in prCMV-SEAP. Thus, there was obtainedthe expression plasmid prCMV-cis#1-SEAP in which cis-element #1 wasinserted between the CMV promoter and the SEAP start methionine codon(FIG. 4A).

Example 5: Activation of Protein Secretion by Cis-Element

Using the cis-element-containing expression plasmid prepared in Example4, investigation was made of the influence on secretion of expressionprotein. The two expression plasmids provided in Example 4, i.e.prCMV-cis#1-SEAP and prCMV-SEAP, were transfected using theLipofectamine LTX reagent (produced by Life Technologies). The cellsused in the transfection were the four cell lines prepared in Example 1.The transfected cells were cultured in a DMEM supplemented with 0.1% FBSfor 20 hours, and then the culture supernatant was mixed with asubstrate solution containing the fluorescent substrate4-Methylumbelliferyl phosphate (4-MUP; produced by Sigma). After thereaction at room temperature for 30 minutes, the mixture was measuredfor fluorescence intensity (excitation at 360 nm, emission at 440 nm)using a fluorophotometer. Also, in order to correct transfectionefficiency by total SEAP cDNA content, total mRNA was extracted fromcell fractions using a Trizol reagent (produced by Life Technologies),and quantitative PCR was performed with SEAP-specific primers toquantitate SEAP cDNA. The SEAP activity ratio of prCMV-cis#1-SEAP toprCMV-SEAP, which was corrected by total SEAP cDNA content, is shown inFIGS. 4B and 4C.

According to the results of this investigation, in the case of using theCHO cells, SEAP activity was increased by 2.7 times by insertion ofcis-element #1. Also in all the cases of using the CHO 3D5 cells, theCHO 5 g cells, and the CHO YA7 cells, SEAP activity was increased by 3.0to 3.2 times by insertion of cis-element #1. These results showed thatin the different types of CHO cells, the amount of protein synthesizedand secreted per transcript can be increased by inserting cis-element #1into an expression unit.

Thereafter, in order to compare the SEAP activity ratios of theaforementioned different types of cells after insertion of cis-element#1 with the SEAP activity of the CHO cells without insertion ofcis-element #1, the SEAP activities of these types of cells uponintroduction of prCMV-cis#1-SEAP were determined, with the SEAP activityof the CHO cells upon introduction of prCMV-SEAP being taken as 1 (FIG.4C). The SEAP activity ratios of the CHO 3D5 cells and the CHO 5 g cellsupon use of cis-element #1 increased by 4.7 and 6.3 times, respectively,as compared to that of the control CHO cells. Also, the SEAP secretoryactivity of the CHO YA7 cells remarkably increased by 9.4 times ascompared to that of the control CHO cells.

Hence, it was demonstrated that the activity for secreting a proteinexpressed from an expression plasmid can be increased by the coexistenceof cis-element #1 and SF3b4 protein or the coexistence of cis-element #1and p180 protein, and that the protein secretory activity of cells canbe more remarkably enhanced by the coexistence of the three factorscis-element #1, SF3b4 protein and p180 protein.

Example 6: Activation of Collagen Secretion by Cis-Element #1

In this example, using a cis-element-containing expression plasmid,investigation was made of the influence on collagen expression.

An expression plasmid for human type I collagen α1 (COL1A1) wasconstructed according to the procedure described below. Morespecifically, a cDNA fragment encoding the full length of COL1A1(GenBank Accession No. NM_000088.3) was inserted and ligated into theNheI-XhoI site of an expression vector for mammalian cells (pEGFP-C3;produced by Clontech) to thereby obtain the cis-element #1-containingand COL1A1 (1 to 5297 nt)-expressing plasmid prCMV-COL1A1 under thecontrol of a CMV promoter. Also, gene fragments each consisting of 127to 4251 nt or 127 to 5297 nt, which encode the full length of COL1A1 ORFalone instead of the full length of the COL1A1 gene, were amplified inthe same way to construct prCMV-COL1A1-ORF and prCMV-COL1A1-ORF-UTR.Further, expression plasmids for human type II collagen α1 (COL2A1) andhuman type III collagen α1 (COL3A1) were constructed according to theprocedure described below. More specifically, a cDNA fragment encodingthe full length of COL2A1 (GenBank Accession No. NP_001835.3) or COL3A1(GenBank Accession No. NP_000081.1) was inserted and ligated into theEcoRV-NotI site of pcDNA-cis#1 in which cis-element #1 was inserted intoan expression vector for mammalian cells (pcDNA; produced byInvitrogen), whereby the COL2A1 (1 to 4464 nt)-expressing plasmidpcDNA-cis#1-COL2A1 or the COL3A1 (1 to 4401 nt)-expressing plasmidpeDNA-cis#1-COL3A1 was obtained under the control of a CMV promoter.

Next, in order to investigate the ability of cis-element #1 to activateprocollagen secretion, prCMV-COL1A1 was transfected by a lipofectionmethod into the three cell lines prepared in Example 1. After thetransfected cells were cultured for 40 hours in a DMEM supplemented with0.1% fetal bovine serum and 200 μM ascorbic acid, COL1A1 procollagencontent in culture supernatant was analyzed by Western blotting (FIG.5A).

According to the results of this analysis, as compared to theprocollagen content in the control CHO cells, which was taken as 1, theprocollagen content in the CHO YA7 cells increased by about 20 times.Also, for the purpose of evaluation of cis-element #1, the cis-element#1-free plasmid prCMV-COL1A1-ORF or prCMV-COL1A1-ORF-UTR was geneticallyintroduced into the control CHO cells in the same way. In these cases,the procollagen contents in culture supernatant were below the limit ofdetection by Western blotting.

Further, in order to investigate the secreted amount ofhomotrimer-forming collagen, pcDNA-cis#1-COL2A1, pcDNA-cis#1-COL3A1, orprCMV-COL1A1 or prCMV-COL1A1-ORF, which were prepared in this example,was transfected by a lipofection method into each of the three celllines prepared in Example 1. After the transfected cells were culturedfor 72 hours in a DMEM supplemented with 2% fetal bovine serum and 200μM ascorbic acid, the culture supernatant was harvested, HCl was addedto 0.1 N to make the pH acidic, pepsin (produced by Sigma) was added to0.5 mg/mL, and digestion reaction was carried out at 4° C. for 16 hours.NaCl was added to the reaction mixture to give a concentration of 1 M,and the mixture was left on ice for 3 hours and then centrifuged. Theresulting precipitate was washed with 1 M NaCl and 95% ethanol. Thethus-obtained purified collagen samples were subjected toelectrophoresis by SDS-PAGE to compare the band intensities of thecollagens. According to the results of this comparison, the amounts ofthe homotrimers COL1A1, COL2A1 and COL3A1 secreted in the CHO YA7 cellssignificantly increased by 1.8, 1.9 and 3.7 times, respectively, ascompared to those in the control CHO cells, which are taken as 1 (FIGS.5B to 5D). Even in the absence of cis-element #1, the homotrimer amountssecreted in the CHO 3D5 cells and the CHO YA7 cells increased by 1.5 and2.1 times, respectively, as compared to the control.

Hence, it was demonstrated that cis-element #1 is capable of enhancingthe expression of collagen molecules in the different types of CHOcells, and that the secreted amount of collagen with maintainedtriple-helical structure is further increased by using cis-element #1 inthe presence of SF3b4 protein and/or p180 protein.

Example 7: Enhancement Effect of Cis-Element on Expression of AntibodyMolecules

The influence of cis-element on antibody expression was investigatedaccording to the procedure described below.

An expression plasmid for full-length antibody heavy and light chainswas constructed according to the procedure described below. Morespecifically, full-length antibody heavy chain (I-IC) and light chain(LC) sequences encoded by the anti-IL-8 antibody-expressing plasmid(p6G425V11N35A.choSD, ATCC 209552) were synthesized by the genesynthesis service (provided by MBL). Then, the full-length heavy chainORF and light chain ORF were inserted and ligated into the NheI-SpeIsite and the KpnI-EcoRV site of the pEF1/Myc-His vector, respectively.Thereafter, the light chain expression cassette was cleaved with ClaI,and inserted and ligated into the ClaI site of the heavy chainexpression vector, whereby the anti-IL-8 antibody (HC, LC) coexpressionplasmid pEF-HC-LC was constructed. Further, cis#1 was inserted upstreamof the heavy chain and light chain ORFs in this plasmid to construct theexpression plasmid pEF-cis#1-HC-LC.

Each of pEF-HC-LC and pEF-cis#1-HC-LC was transfected by a lipofectionmethod into each of the three cell lines prepared in Example 1. Afterthe transfected cells were cultured in a DMEM supplemented with 0.1%fetal bovine serum for 96 hours, antibody production in culturesupernatant was quantified by ELISA using the Human IgG ELISAQuantitation Set (produced by Bethyl). According to the results of thisinvestigation, the secreted antibody amount in the CHO cells wasincreased by 2.7 times by insertion of cis-element #1. Likewise, thesecreted antibody amounts in the CHO 3D5 cells and the CHO YA7 cellswere also increased by 2.5 and 1.8 times, respectively, by insertion ofcis-element #1 (FIG. 6A). These results showed that the secretedantibody amount in the different types of CHO cells can be increased byinserting cis-element #1 into an expression unit.

As shown in FIG. 6B, as compared to the secreted antibody amount in theCHO cells upon introduction of pEF-HC-LC, which was taken as 1, thesecreted antibody amounts in the CHO 3D5 cells incorporating SF3b4 andin the CHO YA7 cells incorporating SF3b4 and p180 increased by 2.5 and12.6 times, respectively, which demonstrated that secreted antibodyamount can be increased by insertion of p180 protein and/or SF3b4protein. These tendencies become more significant when cis-element #1 isused—in the presence of this element, the secreted antibody amounts inthe CHO 3D5 cells and the CHO YA7 cells remarkably increased by 5.6 and21.3 times, respectively, as compared to the control CHO cells.

Hence, it was demonstrated that cis-element #1 acts positively onantibody production, and that the activity of this element becomes moresignificant in the presence of SF3b4 protein or p180 protein, or both ofthese proteins.

Example 8: Comparison Between Kozak Sequence and Cis-Element #1 in Termsof Secretion Activation Effect

This example was intended to compare the secretion activation effect ofa cis-element with that of the kozak sequence which is known as aconsensus sequence involved in the initiation of translation in the mRNAof eukaryocytes.

The expression plasmids prCMV-SEAP-kozak and prCMV-cis#1-SEAP-kozak, inwhich the 6-bp sequence TCCTGC immediately preceding the startmethionine codon ATG of each of prCMV-SEAP and prCMV-cis#1-SEAP preparedin Example 4 was substituted by the sequence GCCACC, were constructedaccording to the procedure described below. More specifically, PCR wasfirst performed with SEAP-specific primers to amplify a SEAP fragment (1to 132 nt) in which the sequence GCCACC was added immediately precedingATG. Then, a SEAP (1 to 132 nt) region was excised from prCMV-SEAP andprCMV-cis#1-SEAP with HindIII-PstI and replaced with the amplifiedfragment treated with HindIII-PstI, whereby there were obtained the SEAPexpression plasmids prCMV-SEAP-kozak and prCMV-cis#1-SEAP-kozak in whichthe kozak sequence was inserted upstream of SEAP ORF.

The plasmids prCMV-SEAP, prCMV-SEAP-kozak, prCMV-cis#1-SEAP, andprCMV-cis#1-SEAP-kozak were each transfected into each of the CHO cells,the CHO 3D5 cells and the CHO YA7 cells, and after the culture for 20hours, SEAP activity in culture supernatant was measured according tothe procedure described in Example 5. The results confirmed that ascompared to the case of prCMV-SEAP, the SEAP activity of the CHO cellswas enhanced by at least twice with prCMV-cis#1-SEAP andprCMV-cis#1-SEAP-kozak (FIG. 7)—the effect of these plasmids iscomparable to or greater than that of the kozak sequence which is knownto be effective for protein expression.

This tendency of enhancement was also strongly observed in the CHO 3D5cells and the CHO YA7 cells. The SLAP activity ratio increased by 3.3 to3.4 times in the presence of cis-element #1, and by 3.3 to 4.9 times inthe presence of both the kozak sequence and cis-element #1 (FIG. 7).

Hence, it was demonstrated that cis-element #1 has a stronger secretionactivation effect on CHO cells, CHO 3D5 cells, and CHO YA7 cells thanthe kozak sequence, and that the secretory activity of these types ofcells can be further enhanced by using the three factors cis-element #1,SF3b4 protein, and p180 protein in combination with the kozak sequence.

Example 9: Protein Expression Enhancement Effect of Addition ofCis-Element

Expression plasmids containing various types of cis-element were eachused in each of the cell lines prepared in Example 1 to investigate thedetails of cis-element sequences with a protein expression enhancementeffect.

The expression vector prCMV-cis#2-SEAP, in which the cis-elementsequence of human-derived fibronectin gene, cis-element #2 (SEQ ID NO:6), was inserted into prCMV-SEAP, was constructed according to theprocedure described below. More specifically, RNA derived from humanfibroblasts was prepared according to the procedure described inNon-patent Literature 1 (Ueno, et al., (2010) J Biol Chem 285,29941-29950), and RT-PCR was performed using the prepared RNA as atemplate. In the process of amplification, a fragment comprisingcis-element #2 was amplified using the primers (SEQ ID NO: 13, SEQ IDNO: 14) in which the BglII and HindIII recognition sequences were addedto sites towards the 5′ and 3′ ends, respectively. The amplifiedfragment was treated with BglII-HindIII, and then inserted and ligatedinto the BglII-HindIII site located between the CMV promoter and SEAPORF in prCMV-SEAP. Thus, there was obtained an expression plasmid inwhich cis-element #2 was inserted between the CMV promoter and the SEAPstart methionine codon.

The expression vectors prCMV-cis#3-SEAP and the like, in which thecis-element sequence of human-derived type I collagen al gene,cis-element #3, or the cis-element sequence of human-derived fibronectingene, cis-element #4, was inserted into prCMV-SEAP, were constructedaccording to the procedure described below. More specifically,cis-element-containing primers designed for cis-element #3 (SEQ ID NO:9, SEQ ID NO: 10) or cis-element-containing primers designed forcis-element #4 (SEQ ID NO: 11, SEQ ID NO: 12) were subjected to heattreatment at 95° C. for 10 minutes and then the temperature was loweredin stages to 25° C. to anneal the two primers, whereby each type oflinker was prepared. These types of linker were each inserted andligated into the BglII-HindIII site located between the CMV promoter andSEAP ORF in prCMV-SEAP. Thus, there were obtained expression plasmids inwhich cis-element #3 (SEQ ID NO: 7) or cis-element #4 (SEQ ID NO: 8) wasinserted between the CMV promoter and the SEAP start methionine codon.

The expression vectors prCMV-cis#5-SEAP and prCMV-cis#6-SEAP in whichcis-element #5 or #6 was inserted into prCMV-SEAP were constructedaccording to the procedure described below. Fragments comprisingcis-element #5 (1-60) or cis-element #6 (61-126) were amplified usingrespective sets of primers (SEQ ID NOs: 28 and 29, or SEQ ID NOs: 30 and31), each having added thereto the partial BglII and HindIII recognitionsequences of cis-element #1. The amplified fragments were each treatedwith BglII-HindIII, and then inserted and ligated into the BglII-HindIIIsite located between the CMV promoter and SEAP ORF in prCMV-SEAP. Thus,there were obtained expression plasmids in which cis-element #5 orcis-element #6 was inserted between the CMV promoter and the SEAP startmethionine codon.

The expression vector prCMV-cis#7-SEAP, in which cis-element #7 wasinserted into prCMV-SEAP, was constructed according to the proceduredescribed below. A COL2A1 gene-derived sequence was synthesized, andthen inserted and ligated into the BglII-HindIII site located betweenthe CMV promoter and SEAP ORF in prCMV-SEAP, using the BglII and HindIIIrecognition sequences added to the ends. Thus, there was obtained anexpression plasmid in which cis-element #7 was inserted between the CMVpromoter and the SEAP start methionine codon.

The expression vectors prCMV-cis#8-SEAP, prCMV-cis#9-SEAP, andprCMV-cis#10-SEAP, in which a cis-element #2-derivedsequence—cis-element #8, #9 or #10—was inserted into prCMV-SEAP, wereconstructed according to the procedure described below. Morespecifically, cis-element-containing primers designed for cis-element #8(SEQ ID NO: 32, SEQ ID NO: 33), cis-element-containing primers designedfor cis-element #9 (SEQ ID NO: 34, SEQ ID NO: 35), orcis-element-containing primers designed for cis-element #10 (SEQ ID NO:36, SEQ ID NO: 37) were subjected to heat treatment at 95° C. for 10minutes and then the temperature was lowered in stages to 25° C. toanneal the two primers, whereby each type of linker was prepared. Thesetypes of linker were each inserted and ligated into the BglII-HindIIIsite located between the CMV promoter and SEAP ORF in prCMV-SEAP. Thus,there were obtained expression plasmids in which cis-element #8 (SEQ IDNO: 24), cis-element #9 (SEQ ID NO: 25), or cis-element #10 (SEQ ID NO:26) was inserted between the CMV promoter and the SEAP start methioninecodon.

The expression vector prCMV-cis#11-SEAP, in which cis-element #11 wasinserted into prCMV-SEAP, was constructed according to the proceduredescribed below. A fragment comprising cis-element #11 (1-113) wasamplified using primers (SEQ ID NO: 38, SEQ ID NO: 39) having addedthereto the partial BglII and HindIII recognition sequences ofcis-element #1. The amplified fragment was treated with BglII-HindIII,and then inserted and ligated into the BglII-HindIII site locatedbetween the CMV promoter and SEAP ORF in prCMV-SEAP. Thus, there wasobtained an expression plasmid in which cis-element #11 (SEQ ID NO: 27)was inserted between the CMV promoter and the SEAP start methioninecodon.

TABLE 4 Table 4: List of primers for cis-element amplification SEQTarget ID cis# Primer sequence NO #1 aaaaaaagat cttcgtcgga gcagacg 15 #1aaaaaaaagc ttgtctagac cctagac 16 #2 aaaaaaagat ctgcccgcgc cggctgt 13 #2aaaaaaaagc ttgttgagac ggtggggga 14 #3gatcttcgtc ggagcagacg ggagtttctc cta 9 #3agcttaggag aaactcccgt ctgctccgac gaa 10 #4gatcttctgc atcccttctg tccctccaca 11 #4 agcttgtgga gggacagaag ggatgcagaa12 #5 aaaaaaagat cttcgtcgga gcagacggga gt 28 #5aaaaaaaagc ttctcacact ccgcgtgcct cc 29 #6aaaaaaagat ctgccacgca tgagcggacg ct 30 #6aaaaaaaagc ttgtctagac cctagacatg ta 31 #8gatctcaggg ggaggagagg gaaccccagg 32 cgcgaa #8agctttcgcg cctggggttc cctctcctcc 33 ccctga #9gatctgagcg ggaagagggg acctgcagcc 34 acaactta #9agcttaagtt gtggctgcag gtcccctctt 35 cccgctca #10gatctcaggg ggaggagagg gaaccccagg 36 cgcgagcggg aagaggggac ctgcagccacaactta #10 agcttaagtt gtggctgcag gtcccctctt 37cccgctcgcg cctggggttc cctctcctcc ccctga #11aaaaaaagat cttcgtcgga gcagacggga gt 38 #11aaaaaaaagc ttacatgtag actctttgtg gc 39

The thus-obtained plasmids prCMV-cis#1-SEAP, prCMV-cis#2-SEAP,prCMV-cis#3-SEAP, prCMV-cis#4-SEAP, prCMV-cis#5-SEAP, prCMV-cis#6-SEAP,prCMV-cis#7-SEAP, prCMV-cis#8-SEAP, prCMV-cis#9-SEAP, prCMV-cis#10-SEAP,prCMV-cis#11-SEAP, and prCMV-SEAP were each transfected into the CHO YA7cells, and after the culture for 20 hours, SEAP activity in culturesupernatant was measured according to the procedure described in Example5. As compared to the SEAP activity in the case of using prCMV-SEAP,which was taken as 1, the SEAP secretory activity ratio was increased byabout 2.0 to 3.4 times with all the cis-elements used (FIG. 8A). In thisprocess, the SF3b4 protein content in membrane fraction was analyzed byWestern blotting and quantified by densitometry; then, it was found thatin the cases of using cis-element #1, cis-element #2, cis-element #3,cis-element #4, cis-element #5, and cis-element #6, the SF3b4 proteincontent in membrane fraction increased significantly by 2.0 to 3.6 timesas compared to the control (FIG. 8B).

These results showed that SF3b4 protein with a protein expressionenhancement effect can be localized onto an endoplasmic reticulummembrane by adding each of cis-elements #1 to #11 to an expression unit,and that the secretory capacity in cells can be enhanced through thislocalization process.

Example 10: Effect of the Chain Length of Intra-Motif Sequence

The motif sequence GAN₁-(X)_(n)-ACN₂ identified in cis-element #1 wasinvestigated for an effective chain length n by the following procedure.Different cis-element #3-derived variants of GAG-(X)_(n)-ACV (Vrepresents A, G or C) with n being varied from 1 to 9(mer) wereconstructed according to the same procedure as in Example 8 (FIG. 9A).Also constructed was a motif-deficient variant (motif delete). For thepurpose of evaluating the SEAP secretory activities of the differentelements, analysis of their secretion activities was made according tothe procedure described in Example 5. According to the results of thisanalysis, a comparable activity to that of cis-element #3 was obtainedin the cases of n=3 to 6 (FIG. 9B). Hence, it was found that the chain-Xlength n of the motif GAN₁-(X)_(n)-ACN₂ in a cis-element, which plays animportant role in the activation of secretion in this system, rangesfrom 3 to 6 residues.

Example 11: Influences of Nucleotide Substitutions/Insertions in MotifSequence

Investigation was made of the influences of nucleotide substitutions andinsertions in a motif on expression enhancement activity. Differentcis-element #3-derived variants composed of the motif GAN₁-(X)_(n)-ACN₂in cis-element #3, wherein N₁ and N₂ are independently A, G, C or T,were constructed by the same procedure as in Example 8 (FIG. 10A). Alsoconstructed were other variants in which the motif was inserted into thepolyA sequence or the polyC sequence, as well as control variants (FIG.10A). The secretory activities of the different elements were analyzedaccording to the procedure described in Example 5. According to theresults of this analysis, a comparable SEAP activity to that ofcis-element #3 was obtained in all the cases where N₂ in cis-element #3was substituted from G to A or C or T (FIG. 10B). Likewise, a comparableSEAP activity to that of cis-element #3 was obtained in all the caseswhere N₁ in cis-element #3 was substituted from G to A or C or T (FIG.10B). Furthermore, the element having the motif inserted thereinto alsohad a comparable activity to that of cis-element #3. Hence, it wasdemonstrated that the motif having high activity is GAN₁-(X)_(n)-ACN₂(wherein N₁ and N₂ can be independently any of the nucleotides A, T, Cand G, and n is an integer of 3 to 6).

Example 12: Decrease in Collagen Secretion in Association withExpression Suppression of SF3b4

Investigation was made of the influence of SF3b4 expression suppressionon collagen secretion. First, siRNA directed against SF3b4 (LifeTechnologies, human SF3b4 siRNA HSS115684) was transfected into humanembryonic lung (HEL) fibroblasts according to the standard protocol forthe Oligofectamine reagent (produced by Life Technologies). Thetransfected cells were cultured for 4 days under the condition of DMEMsupplemented with 0.1% FBS and 200 μM ascorbic acid phosphate ester.Then, the medium was harvested, and the COL1A1 procollagen content inculture supernatant and the SF3b4 protein content in cell fractions wereanalyzed according to the procedure described in Example 6. According tothe results of this analysis, when the amount of SF3b4 expressedintracellularly decreased to 20% of the control, the secreted COL1A1procollagen amount decreased to 10% (FIG. 11). Hence, it wasdemonstrated that collagen production is remarkably suppressed throughsuppression of SF3b4 expression.

Example 13: Influence of a p180/SF3b4-Coexpressing Suspension Cell Lineon Collagen Secretion

A CHO-S cell line stably coexpressing human p180 protein and human SF3b4protein was established according to the procedure described below.First, pCDNA-p180/54R was transfected into CHO-S cells (produced by LifeTechnologies) by a lipofection method, and the transfected cells werecultured for 14 days in the presence of 300 μg/mL of zeocin, wherebydrug selection was done. After zeocin-resistant cell line colonies wereisolated, pEF-SF3b4 was transfected by a lipofection method, and drugselection was done under the condition of 600 μg/mL hygromycin. Afterthe culture for 14 days, cell line colonies resistant to both zeocin andhygromycin were isolated to establish the CHO-S-derived cell line 54#160which stably coexpresses human p180 protein and human SF3b4 protein.

The plasmids prCMV-COL1A1 and prCMV-COL1A1-ORF were each transfected bya lipofection method into each of the control CHO-S cells and theprepared 54#160 cells. The transfected cells were cultured for 96 hoursin a serum-free CD FortiCHO medium supplemented with 8 mM L-glutamine(Life Technologies), and then the COL1A1 procollagen content in culturesupernatant was analyzed by Western blotting. In the case of geneticintroduction of prCMV-COL1A1 containing cis-element #1, the procollagencontent in the 54#160 cells increased by about 3.5 times as compared tothat in the control CHO-S cells, which was taken as 1. In the case ofgenetic introduction of prCMV-COL1A1-ORF not containing cis-element #1,the procollagen content in culture supernatant was below the limit ofdetection by Western blotting.

Hence, it was demonstrated that cis-element #1 is capable of enhancingthe synthesis/secretion of collagen macromolecules in suspended CHO-Scells under a serum-free condition, and that the secretory activity ofsuspended CHO cells can be more remarkably enhanced by the three factorscis-element #1, SF3b4 protein and p180 protein.

Example 14: Shift of mRNA Distribution in Polysome Towards HeavierFractions by Cis-Element #1

The plasmids prCMV-COL1A1 and prCMV-COL1A1-ORF were each transfected bya lipofection method into the CHO YA7 cells. After 40 hours, respectivemembrane fractions were prepared according to the procedure described inExample 3. The resultant membrane fractions were subjected tocentrifugation with a sucrose density gradient from 15 to 50% sucrose tofractionate them into a polysome fraction. mRNA was extracted from eachof the resultant polysome fractions according to the procedure describedin Example 5, and COL1A1 cDNA was quantified by quantitative PCR. Also,the amount of procollagen then secreted was analyzed by the proceduredescribed in Example 6. According to the results of this analysis, theCOL1A1 cDNA distribution in polysome fraction showed a peak at fraction24 in the case of prCMV-COL1A1-ORF not containing a cis-element, and atfraction 26 in the presence of cis#1, which indicated a shift of thedistribution towards heavier fractions (FIG. 12). In addition, with theshift of peak, the secreted procollagen amount increased by 4.9 timesover the control in the presence of cis-element #1. It was demonstratedthat cis-element #1, in the presence of p180 and SF3b4, shows acapability of inducing a shift of mRNA distribution towards heavierfractions, which correlates with an increase in expression.

INDUSTRIAL APPLICABILITY

It was found that, in the recombinant cell of the present inventionwhich has enhanced expression of the full length or a portion of p180protein and the full length or a portion of SF3b4 protein, a DNAencoding a protein as a product of interest is transformed, whereby asynthetic or secretory capacity of the protein as a product of interestis dramatically enhanced.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1: Nucleotide sequence encoding human p180

SEQ ID NO: 2: Amino acid sequence of human p180 protein

SEQ ID NO: 3: Nucleotide sequence encoding human SF3b4

SEQ ID NO: 4: Amino acid sequence of human SF3b4 protein

SEQ ID NO: 5: cis-element #1

SEQ ID NO: 6: cis-element #2

SEQ ID NO: 7: cis-element #3

SEQ ID NO: 8: cis-element #4

SEQ ID NO: 9: cis-element-containing primer designed for cis-element #3

SEQ ID NO: 10: cis-element-containing primer designed for cis-element #3

SEQ ID NO: 11: cis-element-containing primer designed for cis-element #4

SEQ ID NO: 12: cis-element-containing primer designed for cis-element #4

SEQ ID NO: 13: cis-element-containing primer designed for cis-element#2, in which the BglII recognition sequence is added towards the 5′ end

SEQ ID NO: 14: cis-element-containing primer designed for cis-element#2, in which the HindIII recognition sequence is added towards the 3′end

SEQ ID NO: 15: Primer for amplifying cis-element #1, in which the BglIIrecognition sequence is added towards the 5′ end

SEQ ID NO: 16: Primer for amplifying cis-element #1, in which theHindIII recognition sequence is added towards the 3′ end

SEQ ID NO: 17: Motif in cis-element (9mer)

SEQ ID NO: 18: Motif in cis-element (10mer)

SEQ ID NO: 19: Motif in cis-element (11mer)

SEQ ID NO: 20: Motif in cis-element (12mer)

SEQ ID NO: 21: cis-element #5

SEQ ID NO: 22: cis-element #6

SEQ ID NO: 23: cis-element #7

SEQ ID NO: 24: cis-element #8

SEQ ID NO: 25: cis-element #9

SEQ ID NO: 26: cis-element #10

SEQ ID NO: 27: cis-element #11

SEQ ID NO: 28: cis-element-containing primer designed for cis-element#5, in which the BglII recognition sequence is added towards the 5′ end

SEQ ID NO: 29: cis-element-containing primer designed for cis-element#5, in which the HindIII recognition sequence is added towards the 3′end

SEQ ID NO: 30: cis-element-containing primer designed for cis-element#6, in which the BglII recognition sequence is added towards the 5′ end

SEQ ID NO: 31: cis-element-containing primer designed for cis-element#6, in which the HindIII recognition sequence is added towards the 3′end

SEQ ID NO: 32: cis-element-containing primer designed for cis-element #8

SEQ ID NO: 33: cis-element-containing primer designed for cis-element #8

SEQ ID NO: 34: cis-element-containing primer designed for cis-element #9

SEQ ID NO: 35: cis-element-containing primer designed for cis-element #9

SEQ ID NO: 36: cis-element-containing primer designed for cis-element#10

SEQ ID NO: 37: cis-element-containing primer designed for cis-element#10

SEQ ID NO: 38: cis-element-containing primer designed for cis-element#11, in which the BglII recognition sequence is added towards the 5′ end

SEQ ID NO: 39: cis-element-containing primer designed for cis-element#11, in which the HindIII recognition sequence is added towards the 3′end.

The invention claimed is:
 1. A method for increasing an amount of aprotein product of interest expressed in a cell expression systemcomprising inserting a cis-element recognized by or interacting with anRNA-binding protein in an expression unit for expressing the proteinproduct of interest, wherein the expression unit comprises a cDNAencoding a protein product of interest consisting of a nucleic acidsequence encoding the full-length of the protein product of interest,wherein the cis-element is inserted downstream of a promoter andupstream of a start codon of the cDNA encoding the protein product ofinterest, whereby the amount of the protein product of interestexpressed in the cell expression system is increased, wherein thecis-element comprises two or more motifs consisting of a nucleotidesequence containing 9mer to 12mer sequence of GAN1-(X)n-ACN2, whereinn=3 to 6, N1 and N2 are independently selected from adenine (A), thymine(T), cytosine (C), or guanine (G), and X is selected from adenine (A),thymine (T), cytosine (C), or guanine (G), wherein the RNA-bindingprotein is (1) a protein consisting of an amino acid sequence ofsplicing factor 3B subunit 4 (SF3b4) set forth in SEQ ID NO: 4; (2) aprotein consisting of an amino acid sequence with at least 90% sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 4, andwhich, when expressed in the cell, has an ability to enhance capacity ofthe cell for synthesis or secretion of the protein product of interest,wherein the ability is comparable to that of SF3b4 having the amino acidsequence set forth in SEQ ID NO: 4; or (3) a protein consisting of anamino acid sequence encoded by a nucleotide sequence with at least 90%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3,and which, when expressed in the cell, has an ability to enhancecapacity of the cell for synthesis or secretion of the protein productof interest, wherein the ability is comparable to that of SF3b4 havingthe amino acid sequence set forth in SEQ ID NO: 4; and wherein the cellexpression system is a recombinant cell transfected with a nucleic acidencoding an RNA-binding protein containing an RNA recognition motif(RRM) and a nucleic acid encoding a p180 protein, wherein saidtransfection provides for enhanced intracellular synthetic or secretorycapacity of a protein product of interest.
 2. The method according toclaim 1, wherein the cis-element comprises (i) a nucleotide sequencecontaining 9mer to 12mer sequence motifs of GAG-(X)n-ACV, wherein n=3 to6 and V is A, G, or C; and (ii) a nucleotide sequence set forth in oneof SEQ ID NOs: 17 to
 20. 3. The method according to claim 1, wherein thecis-element comprises a sequence selected from the group consisting of:a sequence derived from the nucleotide sequence of the 5′ untranslatedregion of a type I collagen gene; a sequence derived from the nucleotidesequence of the 5′ untranslated region of a fibronectin gene; a sequencederived from the nucleotide sequence of the 5′ untranslated region of amatrix metalloproteinase 14 (MMP14) gene; and a sequence derived fromthe nucleotide sequence of the 5′ untranslated region of a prolyl4-hydroxylase A2 (P4HA2) gene.
 4. The method according to claim 1,wherein the cis-element comprises SEQ ID NO: 5, or nucleotides atpositions 1 to 102, positions 1 to 78, positions 1 to 60, positions 61to 126, positions 16 to 57, positions 79 to 126, positions 103 to 126,positions 58 to 78, positions 51 to 78, positions 1 to 27, or positions70 to 78 of SEQ ID NO:
 5. 5. The method according to claim 1, whereinthe cell expression system comprises a cis-element comprising four ofthe motifs.
 6. A method for increasing an amount of a protein product ofinterest expressed in a cell expression system comprising: (a) insertinga cis-element recognized by or interacting with an RNA-binding proteinin an expression unit for expressing the protein product of interest,wherein the cis-element is inserted downstream of a promoter andupstream of a start codon in a nucleotide sequence encoding the proteinproduct of interest, whereby the amount of the protein product ofinterest expressed in the cell expression system is increased, whereinthe cis-element comprises one or more motif(s) consisting of anucleotide sequence containing 9mer to 12mer sequence of GAN1-(X)n-ACN2,wherein n=3 to 6, N1 and N2 are independently selected from adenine (A),thymine (T), cytosine (C), or guanine (G), and X is selected fromadenine (A), thymine (T), cytosine (C), or guanine (G), and (b)expressing the protein product of interest in the cell expressionsystem, wherein the cell expression system is a recombinant celltransfected with a nucleic acid encoding an RNA-binding proteincontaining an RNA recognition motif (RRM) and transfected with both anucleic acid encoding a p180 protein, wherein the RNA-binding protein is(1) a protein consisting of an amino acid sequence of splicing factor 3Bsubunit 4 (SF3b4) set forth in SEQ ID NO: 4; (2) a protein consisting ofan amino acid sequence with at least 90% sequence identity to the aminoacid sequence set forth in SEQ ID NO: 4, and which, when expressed inthe cell, has an ability to enhance capacity of the cell for synthesisor secretion of the protein product of interest, wherein the ability iscomparable to that of SF3b4 having the amino acid sequence set forth inSEQ ID NO: 4; or (3) a protein consisting of an amino acid sequenceencoded by a nucleotide sequence with at least 90% sequence identity tothe nucleotide sequence set forth in SEQ ID NO: 3, and which, whenexpressed in the cell, has an ability to enhance capacity of the cellfor synthesis or secretion of the protein product of interest, whereinthe ability is comparable to that of SF3b4 having the amino acidsequence set forth in SEQ ID NO:
 4. 7. An expression unit for expressinga protein product of interest comprising (a) a cDNA encoding a proteinproduct of interest consisting of a nucleic acid sequence encoding thefull-length of the protein product of interest, and (b) a cis-elementrecognized by or bound by an RNA-binding protein, wherein thecis-element is inserted into the expression unit downstream of apromoter and and upstream of a start codon of the cDNA encoding theprotein product of interest, whereby the amount of the protein productof interest expressed in a cell expression system is increased, whereinthe cis-element comprises two or more motifs consisting of a nucleotidesequence containing 9mer to 12mer sequence of GAN1-(X)n-ACN2, whereinn=3 to 6, N1 and N2 are independently selected from adenine (A), thymine(T), cytosine (C), or guanine (G), and X is selected from adenine (A),thymine (T), cytosine (C), or guanine (G), wherein the RNA-bindingprotein is (1) a protein consisting of an amino acid sequence ofsplicing factor 3B subunit 4 (SF3b4) set forth in SEQ ID NO: 4; (2) aprotein consisting of an amino acid sequence with at least 90% sequenceidentity to the amino acid sequence set forth in SEQ ID NO:4, and which,when expressed in the cell, has an ability to enhance capacity of thecell for synthesis or secretion of the protein product of interest,wherein the ability is comparable to that of SF3b4 having the amino acidsequence set forth in SEQ ID NO: 4; or (3) a protein consisting of anamino acid sequence encoded by a nucleotide sequence with at least 90%sequence identity to the nucleotide sequence set forth in SEQ ID NO: 3,and which, when expressed in the cell, has an ability to enhancecapacity of the cell for synthesis or secretion of the protein productof interest, wherein the ability is comparable to that of SF3b4 havingthe amino acid sequence set forth in SEQ ID NO: 4; and wherein the cellexpression system is a recombinant cell transfected with a nucleic acidencoding an RNA-binding protein containing an RNA recognition motif(RRM) and a nucleic acid encoding a p180 protein, wherein saidtransfection provides for enhanced intracellular synthetic or secretorycapacity of a protein product of interest.
 8. An expression vectorcomprising the expression unit according to claim
 7. 9. The expressionunit according to claim 7 comprising a cis-element comprising four ofthe motifs.
 10. An expression unit for expressing a protein product ofinterest comprising (a) a cDNA encoding a protein product of interestconsisting of a nucleic acid sequence encoding the full-length of theprotein product of interest, and (b) a cis-element recognized by orbound by an RNA-binding protein, wherein the cis-element is insertedinto the expression unit downstream of a promoter and upstream of astart codon of the cDNA encoding the protein product of interest,whereby the amount of the protein product of interest expressed in acell expression system is increased, wherein the cis-element comprisesone or more motif(s) consisting of a nucleotide sequence containing 9merto 12mer sequence of GAN1-(X)n-ACN2, wherein n=3 to 6, N1 and N2 areindependently selected from adenine (A), thymine (T), cytosine (C), orguanine (G), and X is selected from adenine (A), thymine (T), cytosine(C), or guanine (G), wherein the RNA-binding protein is (1) a proteinconsisting of an amino acid sequence of splicing factor 3B subunit 4(SF3b4) set forth in SEQ ID NO: 4; (2) a protein consisting of an aminoacid sequence with at least 90% sequence identity to the amino acidsequence set forth in SEQ ID NO: 4, and which, when expressed in thecell, has an ability to enhance capacity of the cell for synthesis orsecretion of the protein product of interest, wherein the ability iscomparable to that of SF3b4 having the amino acid sequence set forth inSEQ ID NO: 4; or (3) a protein consisting of an amino acid sequenceencoded by a nucleotide sequence with at least 90% sequence identity tothe nucleotide sequence set forth in SEQ ID NO: 3, and which, whenexpressed in the cell, has an ability to enhance capacity of the cellfor synthesis or secretion of the protein product of interest, whereinthe ability is comparable to that of SF3b4 having the amino acidsequence set forth in SEQ ID NO: 4; wherein the cell expression systemis a recombinant cell transfected with a nucleic acid encoding anRNA-binding protein containing an RNA recognition motif (RRM) andtransfected with both a nucleic acid encoding a p180 protein, whereinsaid transfection provides for enhanced intracellular synthetic orsecretory capacity of a protein product of interest; and wherein theexpression unit comprises a cDNA encoding a protein product of interestconsisting of a nucleic acid sequence encoding the full-length of theprotein product of interest.
 11. An expression vector comprising anexpression unit for expressing a protein product of interest comprising(a) a cDNA encoding a protein product of interest consisting of anucleic acid sequence encoding the full-length of the protein product ofinterest, and (b) a cis-element recognized by or bound by an RNA-bindingprotein, wherein the cis-element is inserted into the expression unitdownstream of a promoter and upstream of a start codon of the cDNAencoding the protein product of interest, whereby the amount of theprotein product of interest expressed in a cell expression system isincreased, wherein the cis-element comprises one or more motif(s)consisting of a nucleotide sequence containing 9mer to 12mer sequence ofGAN1-(X)n-ACN2, wherein n=3 to 6, N1 and N2 are independently selectedfrom adenine (A), thymine (T), cytosine (C), or guanine (G), and X isselected from adenine (A), thymine (T), cytosine (C), or guanine (G),wherein the RNA-binding protein is (1) a protein consisting of an aminoacid sequence of splicing factor 3B subunit 4 (SF3b4) set forth in SEQID NO: 4; (2) a protein consisting of an amino acid sequence with atleast 90% sequence identity to the amino acid sequence set forth in SEQID NO: 4, and which, when expressed in the cell, has an ability toenhance capacity of the cell for synthesis or secretion of the proteinproduct of interest, wherein the ability is comparable to that of SF3b4having the amino acid sequence set forth in SEQ ID NO: 4; or (3) aprotein consisting of an amino acid sequence encoded by a nucleotidesequence with at least 90% sequence identity to the nucleotide sequenceset forth in SEQ ID NO: 3, and which, when expressed in the cell, has anability to enhance capacity of the cell for synthesis or secretion ofthe protein product of interest, wherein the ability is comparable tothat of SF3b4 having the amino acid sequence set forth in SEQ ID NO: 4;wherein the cell expression system is a recombinant cell transfectedwith a nucleic acid encoding an RNA-binding protein containing an RNArecognition motif (RRM) and transfected with both a nucleic acidencoding a p180 protein, wherein said transfection provides for enhancedintracellular synthetic or secretory capacity of a protein product ofinterest; and wherein the expression unit comprises a cDNA encoding aprotein product of interest consisting of a nucleic acid sequenceencoding the full-length of the protein product of interest.